Genetically modified non-human animals with humanized immunoglobulin locus

ABSTRACT

The present disclosure relates to genetically modified animals and cells with humanized heavy chain immunoglobulin locus and/or humanized light chain immunoglobulin locus.

CLAIM OF PRIORITY

This application is a divisional application of U.S. application Ser. No. 17/010,587, filed on Sep. 2, 2020, which is a pursuant to 35 U.S.C. § 119(e) is a continuation of International Application PCT/CN2020/075698, with an international filing date of Feb. 18, 2020, which then claims priority to PCT/CN2019/075406, filed on Feb. 18, 2019 and PCT/CN2019/106320, filed on Sep. 18, 2019 under 35 U.S.C. § 365(b). The entire contents of the foregoing are incorporated herein by reference.

TECHNICAL FIELD

This disclosure relates to genetically modified animals and cells with humanized heavy chain immunoglobulin locus and/or humanized light chain immunoglobulin locus.

SEQUENCE LISTING

This application contains a Sequence Listing that has been submitted electronically as an XML file named “44835-0034002.XML.” The XML file, created on Sep. 20, 2022, is 82,297 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

BACKGROUND

Therapeutic antibodies are one of the fastest growing classes of therapeutic compounds, rapidly outpacing the growth of small-molecule drugs. These therapeutic antibodies are usually human or humanized antibodies. The human or humanized antibodies can be generated by humanization of a rodent antibody (e.g., a mouse antibody) or by using phage libraries. The antibodies that are generated by these methods often have suboptimal binding affinities and biophysical attributes, leading to difficulties in manufacture and poor pharmacokinetics. Particularly, the humanization process may adversely affect the binding affinity and introduce immunogenic epitopes to the antibodies, and antibodies discovered using phage libraries show limited diversity and non-native pairing of immunoglobulin heavy and light chains. Iterative and time-consuming experiments are often required to improve the properties. And in some cases, these antibodies can also be immunogenic in patients, leading to attenuation of their efficacy over time.

One possible approach for generating fully human antibodies is to use transgenic animals engineered to express a human antibody repertoire. The generation of transgenic animals, such as mice having varied immunoglobulin loci, has allowed the use of such transgenic animals in various research and development applications, e.g., in drug discovery and basic research into various biological systems. Many of the early generation transgenic animals had incomplete human antibody repertoires, had antibody production below the normal rates due to less efficient V(D)J recombination, had endogenous antibody repertoires which may introduce immunogenic epitopes, and various other issues. There is a need for efficient and cost-effective methods of producing human antibodies, and a need for non-human animals comprising humanized immunoglobulin locus, which have the ability to respond to an antigen to generate humanized antibodies.

SUMMARY

The present disclosure relates to genetically modified animals and cells with humanized heavy chain and light chain immunoglobulin locus.

In some aspects, the disclosure relates to a genetically-modified, non-human animal comprising at an endogenous heavy chain immunoglobulin gene locus, one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes. In some embodiments, the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operably linked and can undergo VDJ rearrangement.

In some embodiments, the animal comprises about or at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161 human IGHV genes selected from Table 1, about or at least 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes selected from Table 2, and about or at least 5, 6, 7, 8, or 9 human IGHJ genes selected from Table 3. In some embodiments, the animal comprises all human IGHV genes in Table 1 except IGHV2-10, IGHV3-9, and IGHV1-8, all human IGHD genes in Table 2, and all human IGHJ genes in Table 3. In some embodiments, the animal comprises all human IGHV genes in Table 1 except IGHV5-10-1 and IGHV3-64D, all human IGHD genes in Table 2, and all human IGHJ genes in Table 3. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin gene locus of human chromosome 14 of a human subject. In some embodiments, the animal comprises all human IGHV genes, all human IGHD genes, and all human IGHJ genes at the endogenous heavy chain immunoglobulin gene locus of human chromosome 14 of a human cell (e.g., a somatic cell, a cultured cell, a non-immune cell, a cell without any V(D)J rearrangement).

In some embodiments, the animal comprises a disruption in the animal's endogenous heavy chain immunoglobulin gene locus.

In some embodiments, the animal is a mouse and the disruption in the animal's endogenous heavy chain immunoglobulin gene locus comprises a deletion of one or more mouse IGHV genes in Table 4, one or more mouse IGHD genes in Table 5, and/or one or more mouse IGHJ genes in Table 6.

In some embodiments, the animal is a mouse and the disruption in the animal's endogenous heavy chain immunoglobulin gene locus comprises a deletion of a contiguous sequence starting from mouse IGHV1-85 gene to mouse IGHJ4 gene.

In some embodiments, the animal comprises one or more endogenous IGHM, IGHδ, IGHG3, IGHG1, IGHG2b, IGHG2a, IGHE, and IGHA genes.

In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus.

In some embodiments, the unmodified human sequence is about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 kb.

In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHV1-2. In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHV6-1. In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHD1-1 to human IGHJ6. In some embodiments, the animal comprises an unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHJ6.

In some embodiments, the animal is homozygous with respect to the heavy chain immunoglobulin gene locus. In some embodiments, the animal is heterozygous with respect to the heavy chain immunoglobulin gene locus.

In some embodiments, the animal further comprises at an endogenous light chain immunoglobulin gene locus, one or more human IGKV genes, and one or more human IGKJ genes.

In some embodiments, the animal comprises a disruption in the animal's endogenous lambda light chain immunoglobulin gene locus.

In some embodiments, the animal is a rodent (e.g., a mouse).

In some aspects, the disclosure relates to a genetically-modified animal comprising at an endogenous heavy chain immunoglobulin gene locus, a first sequence comprising one or more human IGHV genes; a second sequence comprising an endogenous sequence; and a third sequence comprising one or more human IGHD genes, and one or more human IGHJ genes, wherein the first sequence, the second sequence, and the third sequence are operably linked.

In some embodiments, the first sequence comprises about or at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161 human IGHV genes selected from Table 1. In some embodiments, the first sequence comprises about or at least 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes selected from Table 2.

In some embodiments, the first sequence is an unmodified sequence derived from a human heavy chain immunoglobulin gene locus. In some embodiments, the first sequence is about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 kb.

In some embodiments, the second sequence comprises an endogenous sequence that is about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 kb.

In some embodiments, the third sequence comprises about or at least 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes selected from Table 2. In some embodiments, the third sequence comprises about or at least 5, 6, 7, 8, or 9 human IGHJ genes selected from Table 3. In some embodiments, the third sequence comprises all human IGHD genes in Table 2, and all human IGHJ genes in Table 3.

In some embodiments, the third sequence is an unmodified sequence derived from a human heavy chain immunoglobulin gene locus. In some embodiments, the third sequence is about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 kb.

In some embodiments, the animal comprises a disruption in the animal's endogenous heavy chain immunoglobulin gene locus.

In some embodiments, the animal is a mouse and the disruption in the animal's endogenous heavy chain immunoglobulin gene locus comprises a deletion of one or more mouse IGHV genes in Table 4, one or more mouse IGHD genes in Table 5, and one or more mouse IGHJ genes in Table 6.

In some embodiments, the animal is a mouse and the disruption in the animal's endogenous heavy chain immunoglobulin gene locus comprises a deletion of a sequence starting from mouse IGHV1-85 to mouse IGHJ4.

In some embodiments, the animal comprises one or more endogenous genes selected from the group consisting of IGHM, IGHδ, IGHG3, IGHG1, IGHG2b, IGHG2a, IGHE, and IGHA genes.

In some embodiments, the animal is homozygous with respect to the heavy chain immunoglobulin gene locus. In some embodiments, the animal is heterozygous with respect to the heavy chain immunoglobulin gene locus.

In some embodiments, the animal further comprises at an endogenous light chain immunoglobulin gene locus, one or more human IGKV genes, and one or more human IGKJ genes.

In some embodiments, the animal comprises a disruption in the animal's endogenous lambda light chain immunoglobulin gene locus.

In some embodiments, the animal is a rodent (e.g., a mouse).

In some aspects, the disclosure relates to a genetically-modified, non-human animal comprising at an endogenous light chain immunoglobulin gene locus, one or more human IGKV genes and one or more human IGKJ genes.

In some embodiments, the animal comprises about or at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 human IGKV genes in Table 7, and/or comprises about or at least 1, 2, 3, 4, or 5 human IGKJ genes in Table 8.

In some embodiments, the animal comprises an unmodified sequence derived from a human light chain immunoglobulin gene locus starting from human IGKV3D-7 to human IGKJ5.

In some embodiments, the animal comprises a disruption in the animal's endogenous light chain immunoglobulin gene locus.

In some embodiments, the animal is a mouse and the disruption in the animal's endogenous light chain immunoglobulin gene locus comprises a deletion of one or more mouse IGKV genes in Table 9 and one or more mouse IGKJ genes in Table 10. In some embodiments, the animal comprises all human IGKV genes, and all human IGKJ genes at the endogenous kappa chain immunoglobulin gene locus of human chromosome 2 of a human subject. In some embodiments, the animal comprises all human IGKV genes, and all human IGKJ genes at the endogenous heavy chain immunoglobulin gene locus of human chromosome 2 of a human cell (e.g., a somatic cell, a cultured cell, a non-immune cell, a cell without any V(D)J rearrangement).

In some embodiments, the animal is a mouse and the disruption in the animal's endogenous light chain immunoglobulin gene locus comprises a deletion of a sequence starting from mouse IGKV2-137 to mouse IGKJ5.

In some embodiments, the animal comprises an endogenous IGKC.

In some embodiments, the animal is homozygous with respect to the light chain immunoglobulin gene locus. In some embodiments, the animal is heterozygous with respect to the light chain immunoglobulin gene locus.

In some embodiments, the animal further comprises at an endogenous heavy chain immunoglobulin gene locus, one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes.

In some embodiments, the animal comprises a disruption in the animal's endogenous lambda light chain immunoglobulin gene locus.

In some embodiments, the animal is a rodent (e.g., a mouse).

In some aspects, the disclosure relates to a genetically-modified, non-human animal whose genome comprises an endogenous heavy chain immunoglobulin locus comprising: a replacement of one or more endogenous IGHV, endogenous IGHD, and endogenous IGHJ genes with one or more human IGHV, human IGHD, and human IGHJ genes. In some embodiments, human IGHV, human IGHD, and human IGHJ genes are operably linked to one or more of endogenous genes selected from the group consisting of IGHM, IGHδ, IGHG, IGHE, and IGHA genes.

In some embodiments, one or more endogenous IGHV, endogenous IGHD, and endogenous IGHJ genes are replaced by about or at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161 human IGHV genes in Table 1, about or at least 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes in Table 2, and about or at least 5, 6, 7, 8, or 9 human IGHJ genes in Table 3.

In some embodiments, the animal is a mouse, and about or at least 180 mouse IGHV genes in Table 4, all mouse IGHD genes in Table 5, and all mouse IGHJ genes in Table 6 are replaced.

In some aspects, the disclosure relates to a genetically-modified, non-human animal whose genome comprises an endogenous light chain immunoglobulin locus comprising: a replacement of one or more endogenous IGKV and endogenous IGKJ genes with one or more human IGKV and human IGKJ genes. In some embodiments, the human IGKV and human IGKJ genes are operably linked to an endogenous IGKC gene.

In some embodiments, one or more endogenous IGKV and endogenous IGKJ genes are replaced by about or at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 human IGKV genes in Table 7, and about or at least 1, 2, 3, 4, or 5 human IGKJ genes in Table 8.

In some embodiments, the animal is a mouse, and all mouse IGKV genes in Table 9 and all mouse IGKJ genes in Table 10 are replaced.

In some embodiments, the animal lacks an endogenous immunoglobulin heavy chain variable region locus that is capable of rearranging and forming a nucleic acid sequence that encodes an endogenous heavy chain variable domain (e.g., a mouse heavy chain variable domain).

In some embodiments, the animal lacks an endogenous immunoglobulin light chain variable region locus that is capable of rearranging and forming a nucleic acid sequence that encodes an endogenous light chain variable domain (e.g., a mouse light chain variable domain).

In some embodiments, the animal can produce a humanized antibody.

In some aspects, the disclosure relates to a cell obtained from the animal as described herein.

In some embodiments, the cell is a B cell that expresses a chimeric immunoglobulin heavy chain comprising an immunoglobulin heavy chain variable domain that is derived from a rearrangement of one or more human IGHV genes, one or more human IGHD genes, and one or more human IGHJ genes. In some embodiments, the immunoglobulin heavy chain variable domain is operably linked to a non-human heavy chain constant region.

In some embodiments, the cell is a B cell that expresses a chimeric immunoglobulin light chain comprising an immunoglobulin light chain variable domain that is derived from a rearrangement of one or more human IGKV genes and one or more human IGKJ genes, and wherein the immunoglobulin light chain variable domain is operably linked to a non-human light chain constant region.

In some embodiments, the cell is an embryonic stem (ES) cell.

In some aspects, the disclosure relates to a method of making a chimeric antibody that specifically binds to an antigen, the method comprising exposing the animal as described herein to the antigen; producing a hybridoma from a cell collected from the animal; and collecting the chimeric antibody produced by the hybridoma. In some embodiments, the cell of interest is isolated and sequencing is performed to determine the sequences of rearranged heavy chain variable region and light chain variable region.

In some embodiments, the method further comprises sequencing the genome of the hybridoma.

In some aspects, the disclosure relates to a method of modifying genome of a cell, the method comprising modifying a human chromosome; introducing the modified human chromosome into a cell of the animal; and inducing recombination between the modified human chromosome and an endogenous chromosome, thereby replacing one or more endogenous genes with one or more human genes.

In some embodiments, the modified human chromosome comprises two or more exogenous recombination sites.

In some embodiments, the endogenous chromosome comprises two or more exogenous recombination sites.

In some embodiments, about or at least 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161 human IGHV genes selected from Table 1, about or at least 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes selected from Table 2, and about or at least 5, 6, 7, 8, or 9 human IGHJ genes selected from Table 3 are integrated into the endogenous chromosome by recombination.

In some embodiments, about or at least 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 human IGKV genes in Table 7, and about or at least 1, 2, 3, 4, or 5 human IGKJ genes in Table 8 are integrated into the endogenous chromosome by recombination.

In some embodiments, a human sequence is integrated into the endogenous chromosome by recombination, and the human sequence is about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 kb.

In one aspect, the disclosure provides a method of making an antibody that specifically binds to an antigen. The method involves obtaining a nucleic acid sequence encoding human heavy and light chain immunoglobulin variable regions in a cell that expresses a hybrid antibody that specifically binds to the antigen, wherein the cell is obtained by exposing the animal as described herein to the antigen; operably linking the nucleic acid encoding the human heavy chain immunoglobulin variable region with a nucleic acid encoding a human heavy chain immunoglobulin constant region and the nucleic acid encoding the human light chain immunoglobulin variable region with a nucleic acid encoding a human light chain immunoglobulin constant region; and expressing the nucleic acid in a cell, thereby obtaining the antibody.

In one aspect, the disclosure provides a method of obtaining a nucleic acid that encodes an antibody binding domain that specifically binds to an antigen. The method involves exposing the animal as described herein to the antigen; and sequencing nucleic acids encoding human heavy and light chain immunoglobulin variable regions in a cell that expresses a hybrid antibody that specifically binds to the antigen.

In one aspect, the disclosure provides a method of obtaining a sample, the method comprising exposing the animal as described herein to the antigen; and collecting the sample from the animal. In some embodiments, the sample is a spleen tissue, a spleen cell, or a B cell.

In one aspect, the disclosure provides a method of making an antibody that specifically binds to an antigen. The method involves exposing the animal as described herein to the antigen; obtaining the sequence of (e.g. by sequencing) nucleic acids encoding human heavy and light chain immunoglobulin variable regions in a cell that expresses a hybrid antibody that specifically binds to the antigen; and operably linking in a cell the nucleic acid encoding the human heavy chain immunoglobulin variable region with a nucleic acid encoding a human heavy chain immunoglobulin constant region and the nucleic acid encoding the human light chain immunoglobulin variable region with a nucleic acid encoding a human light chain immunoglobulin constant region.

The disclosure also relates to an offspring of the non-human mammal.

In some embodiments, the non-human mammal is a rodent. In some embodiments, the non-human mammal is a mouse.

The disclosure also provides to a cell including the targeting vector as described herein.

The disclosure also relates to a cell (e.g., a stem cell, an embryonic stem cell, an immune cell, a B cell, a T cell, or a hybridoma) or a cell line, or a primary cell culture thereof derived from the non-human mammal or an offspring thereof. The disclosure further relates to the tissue, organ or a culture thereof derived from the non-human mammal or an offspring thereof.

The disclosure further relates to the use of the non-human mammal or an offspring thereof, the animal model generated through the method as described herein in the development of a product related to an immunization processes, the manufacture of a human antibody, or the model system for a research in pharmacology, immunology, microbiology and medicine.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1A is a flow chart of a method of introducing human immunoglobulin genes into the mouse genome.

FIG. 1B is an overview of replacing mouse immunoglobulin heavy chain variable region with human immunoglobulin heavy chain variable region.

FIG. 1C is an overview of replacing mouse immunoglobulin light chain variable region with human immunoglobulin light chain variable region.

FIG. 2 is a schematic diagram showing the mouse heavy chain immunoglobulin locus.

FIG. 3A is a schematic diagram showing the mouse heavy chain immunoglobulin locus after two recombination sites were introduced to the genome.

FIG. 3B is a schematic diagram showing the mouse heavy chain immunoglobulin locus after recombination with a targeting vector.

FIG. 4 shows a targeting strategy for modifying the mouse heavy chain immunoglobulin locus.

FIG. 5A shows PCR assay results using the mIgHV-5′loxP-L-GT-F/mIGHV-005-L-GT-R2 primer pair.

FIG. 5B shows PCR assay results using the mIGHV-005-5′loxP-R-GT-F2/mIgHV-5′loxP-R-GT-R primer pair.

FIG. 6A shows PCR assay results using the mIGHV-3′lox-L-GT-F2/mIGHV-3′lox-L-GT-R2 primer pair.

FIG. 6B shows PCR assay results using the mIGHV3′lox-R-GT-F2/mIGHV-3′lox-R-GT-R1 primer pair.

FIG. 7 is a schematic diagram of the human chromosome 14 highlighting the heavy chain immunoglobulin locus (not drawn to scale). The heavy chain immunoglobulin locus has the variable regions (V_(H), D_(H), J_(H)) and the constant region (C_(H)). V_(H) represents the segment for the IGHV gene cluster, D_(H) represents the segment for the IGHD gene cluster, J_(H) represents the segment for the IGHJ gene cluster, and C_(H) represents the gene cluster that express constant domains.

FIG. 8 is a schematic diagram showing the human chromosome 14 after the modification.

FIG. 9 shows modifications on the human chromosome 14 with two vectors.

FIG. 10 shows modified human chromosome 14.

FIG. 11 shows the results of PCR assays for loxP site 301 on chromosome hChr14-mut3 using the hIGHV-5′loxP-L-GT-F1 and hIGHV-5′ loxP-R-GT-R primer pair. 8-D7 is a positive control clone.

FIG. 12 shows the results of PCR assays for loxP site 302 on chromosome hChr14-mut3. 8-D7 is a negative control clone.

FIG. 13 is a fluorescence in situ hybridization (FISH) image of cells before the human chromosome 14 is modified.

FIG. 14 is a FISH image of cells after the human chromosome 14 is modified.

FIG. 15 is a schematic diagram showing the modified mouse chromosome 12.

FIG. 16 is a schematic diagram showing Cre mediated recombination, which replaces mouse heavy chain variable region locus with the corresponding human genomic DNA sequence.

FIG. 17 shows PCR assay results using the M5-L primer pair and the M5-R primer pair.

FIG. 18 shows PCR assay results using the M3 primer pair.

FIG. 19 shows PCR assay results using the H5-L primer pair and the H5-R primer pair.

FIG. 20 shows PCR assay results using the H3-L primer pair and the H3-R primer pair.

FIG. 21 is a FISH image. The white arrows (1) and (2) indicate mouse chromosome 12. The white arrow (3) indicates human chromosome fragment labeled by human-specific IGH Breakapart probe.

FIG. 22 is a schematic diagram showing the Flp-mediated recombination.

FIG. 23 is a schematic diagram showing the mouse light chain immunoglobulin locus.

FIG. 24A is a schematic diagram showing the mouse light chain immunoglobulin locus after two recombination sites were introduced to the genome.

FIG. 24B is a schematic diagram showing the mouse light chain immunoglobulin locus after recombination with a targeting vector.

FIG. 25 is a schematic diagram showing a gene targeting strategy for mouse chromosome 6.

FIG. 26 shows PCR assay results using the IGKV-005-C-5G-L-GT-F/IGKV-005-C-5G-L-GT-R2 primer pair.

FIG. 27 shows PCR assay results using the IGKV-005-C-5G-R-GT-F1/IGKV-005-C-5G-R-GT-R primer pair.

FIG. 28 is a schematic diagram of the human chromosome 2 highlighting the light chain immunoglobulin locus (not drawn to scale). V_(HK) represents the segment for the IGKV gene cluster, J_(HK) represents the segment for the IGKJ gene cluster, and C_(HK) represents the IGKC gene.

FIG. 29 is a schematic diagram showing the modified human chromosome 2.

FIG. 30 is a schematic diagram showing a gene targeting strategy for human chromosome 2.

FIG. 31 shows PCR assay results after the first recombination (introducing the vector 2702). WT is the wildtype H9 cells.

FIG. 32 is a FISH image result. The white arrow indicates the modified human chromosome 2 with the correct recombination.

FIG. 33 shows PCR assay results after the second recombination (introducing the vector 2701).

FIG. 34 is a schematic diagram showing Cre mediated recombination, in which human light chain variable region genomic DNA sequence was added to the corresponding mouse locus.

FIG. 35 is a fluorescence in situ hybridization (FISH) image. The arrows (1) and (3) indicate mouse chromosome 6. The arrow (2) indicates human chromosome fragment labeled by human-specific IGK Breakapart probe.

FIG. 36 is a schematic diagram showing the Flp-mediated recombination.

FIG. 37 is a schematic diagram showing human immunoglobulin heavy chain (IGH) locus on chromosome 14 (14q32.33).

FIG. 38 is a schematic diagram showing mouse (Mus musculus) IGH locus on chromosome 12 (12F2) (strain C57BL/6).

FIG. 39 is a schematic diagram showing human immunoglobulin kappa chain (IGK) locus on chromosome 2 (2p11.2).

FIG. 40 is a schematic diagram showing mouse (Mus musculus) IGK locus on chromosome 6 (6C1).

FIG. 41 lists IMGT repertoire for human heavy chain immunoglobulin locus (IGH).

FIG. 42 lists IMGT repertoire for mouse IGH.

FIG. 43 lists IMGT repertoire for human kappa chain immunoglobulin locus (IGK).

FIG. 44 lists IMGT repertoire for mouse IGK.

FIG. 45 shows percentages of leukocytes detected in peripheral blood.

FIG. 46 shows percentages of leukocytes detected in spleen cells.

FIG. 47 shows percentages of leukocytes detected in lymph nodes.

FIG. 48 shows percentages of splenic B cells at different developmental stages.

FIG. 49 shows percentages of lymph nodes B cells at different development stages.

FIG. 50 shows percentages of splenic B cells at spleen marginal zone (MZ-B) and follicular zone (FO-B).

FIG. 51A shows flow cytometry analysis results for B cells at different development stages in the bone marrow obtained from wild-type mice. Area 1 represents pro-B-cells, Area 2 represents pre-B-cells, and Area 3 represents immature B-cells.

FIG. 51B shows flow cytometry analysis results for B cells at different development stages in the bone marrow obtained from humanized heavy chain heterozygous mice. Area 1 represents pro-B-cells, Area 2 represents pre-B-cells, and Area 3 represents immature B-cells.

FIG. 51C shows flow cytometry analysis results for B cells at different development stages in the bone marrow obtained from humanized heavy chain homozygous mice. Area 1 represents pro-B-cells, Area 2 represents pre-B-cells, and Area 3 represents immature B-cells.

FIG. 52 shows IgA isotype levels in serially diluted mouse serum.

FIG. 53 shows IgG1 isotype levels in serially diluted mouse serum.

FIG. 54 shows IgG2b isotype levels in serially diluted mouse serum.

FIG. 55 shows IgG2c isotype levels in serially diluted mouse serum.

FIG. 56 shows IgG3 isotype levels in serially diluted mouse serum.

FIG. 57 shows IgM isotype levels in serially diluted mouse serum.

FIG. 58 shows distribution of the detected IGKV gene expression after VJ recombination among individual mice.

FIG. 59 shows flow cytometry results of wild-type mice after being immunized by human BTLA.

FIG. 60 shows flow cytometry results of humanized heavy chain homozygous mice after being immunized by human BTLA.

FIG. 61 shows flow cytometry results of wild-type mice after being immunized by canine PD-1 (dPD-1).

FIG. 62 shows flow cytometry results of humanized heavy chain homozygous mice after being immunized by canine PD-1 (dPD-1).

FIG. 63 shows a summary of ELISA results of wildtype mice (black bars; mice were labeled with 1-5) and humanized heavy chain homozygous mice (gray bars; mice were labeled with 6-10) after being immunized by ovalbumin (OVA).

FIG. 64 shows a list of human distal Vκ cluster IGKV genes and a list of human proximal Vκ cluster IGKV genes.

FIG. 65A shows body weight of naïve wild-type mice and hVH/hVL mice.

FIG. 65B shows spleen weight of naïve wild-type mice and hVH/hVL.

FIG. 66 shows percentage of immune cells in the spleen of naïve wild-type mice and hVH/hVL mice.

FIG. 67A shows percentage of transitional type 1 (T1, B220⁺IgM⁺IgD⁻), transitional type 2 (T2, B220⁺IgM⁺IgD⁺) and mature (M, B220⁺IgM^(low)IgD⁺) B cell population in spleen B cells. The spleen B cells are from naïve wild-type or hVH/hVL mice.

FIG. 67B shows percentage of marginal-zone (MZ) and follicular (FO) B cell population in spleen B cells. The spleen B cells are from naïve wild-type or hVH/hVL mice.

FIG. 68A shows percentage of pro-B-cell (B220^(low)CD43^(high)IgM^(low)), pre-B-cell (B220^(low)CD43^(int)IgM^(low)) and immature-B-cell (B220^(high)CD43^(low)IgM^(high)) population in bone marrow B cells. The bone marrow B cells are from naïve wild-type or hVH/hVL mice.

FIG. 68B shows percentage of plasma cell (B220^(low)IgM⁻IgD⁻CD138⁻) and memory B cell (B220⁺IgM⁺IgD⁻CD38⁺) population in bone marrow B cells. The bone marrow B cells are from naïve wild-type or hVH/hVL mice.

FIG. 68C shows percentage of plasma cell (B220^(low)IgM⁻IgD⁻CD138⁻) and memory B cell (B220⁺IgM⁺IgD⁻CD38⁺) population in spleen B cells. The spleen B cells are from naïve wild-type or hVH/hVL mice.

FIG. 69 shows concentration of serum immunoglobulin (Ig) subtypes in naïve wild-type or hVH/hVL mice. The Ig subtype concentrations were quantitatively measured by ELISA.

FIG. 70A shows IGHV usage (frequency >1%) in naïve hVH/hVL mice.

FIG. 70B shows IGHV usage (frequency <1%) in naïve hVH/hVL mice.

FIG. 70C shows IGHD usage in naïve hVH/hVL mice.

FIG. 70D shows IGHJ usage in naïve hVH/hVL mice.

FIG. 71A shows IGKV usage (frequency >1%) in naïve hVH/hVL mice.

FIG. 71B shows IGKV usage (frequency <1%) in naïve hVH/hVL mice.

FIG. 71C shows IGKJ usage in naïve hVH/hVL mice.

FIG. 72 is a histogram showing heavy chain CDR3 amino acid length distribution from naïve hVH/hVL mice.

FIG. 73 shows amino acid frequency at heavy chain CDR3 in naïve hVH/hVL mice.

FIG. 74 shows the frequency of HCDR3 that contains cysteine residues in the hVH/hVL mice.

FIG. 75A is a histology image of spleen from naïve wild-type mice.

FIG. 75B is a histology image of inguinal lymph node from naïve wild-type mice.

FIG. 75C is a histology image of Peyer's patch from naïve wild-type mice.

FIG. 75D is a histology image of spleen from naïve hVH/hVL mice.

FIG. 75E is a histology image of inguinal lymph node from naïve hVH/hVL mice.

FIG. 75F is a histology image of Peyer's patch from naïve hVH/hVL mice.

FIG. 76A shows BCMA-specific antibody titer post second and third immunization using human BCMA (B-cell maturation antigen) as antigens in wild-type and hVH/hVL mice.

FIG. 76B shows IL4R-specific antibody titer post second and third immunization using human IL4R (interleukin-4 receptor) as antigens in wild-type and hVH/hVL mice.

FIG. 76C shows PD-1-specific antibody titer post second and third immunization using human PD-1 (Programmed cell death protein 1) as antigens in wild-type and hVH/hVL mice.

FIG. 76D shows Siglec15-specific antibody titer post second and third immunization using human Siglec15 (sialic acid binding ig-like lectin 15) as antigens in wild-type and hVH/hVL mice.

FIG. 76E shows SIRPα-specific antibody titer post second and third immunization using human SIRPα (signal regulatory protein a) as antigens in wild-type and hVH/hVL mice.

FIG. 77A shows body weight of wild-type mice and hVH/hVL mice after immunization.

FIG. 77B shows spleen weight of wild-type mice and hVH/hVL mice after immunization.

FIG. 78 shows percentage of immune cells in the spleen of wild-type mice and hVH/hVL mice after immunization.

FIG. 79A shows percentage of transitional type 1 (T1, B220⁺IgM⁺IgD⁻), transitional type 2 (T2, B220⁺IgM⁺IgD⁺) and mature (M, B220⁺IgM^(low)IgD⁺) B cell population in spleen B cells. The spleen B cells are from wild-type or hVH/hVL mice after immunization.

FIG. 79B shows percentage of marginal-zone (MZ) and follicular (FO) B cell population in spleen B cells. The spleen B cells are from wild-type or hVH/hVL mice after immunization.

FIG. 80A shows percentage of Pro-B-cell (B220^(low)CD43^(high) IgM^(low)), Pre-B-cell (B220^(low)CD43^(int)IgM^(low)) and immature-B-cell (B220^(high)CD43^(low)IgM^(high)) population in bone marrow B cells. The bone marrow B cells are from wild-type or hVH/hVL mice after immunization.

FIG. 80B shows percentage of plasma cell (B220^(low)IgM⁻IgD⁻CD138⁻) and memory B cell (B220⁺IgM⁺IgD⁻CD38⁺) population in bone marrow B cells. The bone marrow B cells are from wild-type or hVH/hVL mice after immunization.

FIG. 80C shows percentage of plasma cell (B220^(low)IgM⁻IgD⁻CD138⁻) and memory B cell (B220⁺IgM⁺IgD⁻CD38⁺) population in spleen B cells. The spleen B cells are from wild-type or hVH/hVL mice after immunization.

FIG. 81 shows concentration of serum immunoglobulin (Ig) subtypes in naïve wild-type or hVH/hVL mice or after the third immunization. The Ig subtype concentrations were determined by ELISA.

FIG. 82 shows serum total IgG concentration in naïve wild-type or hVH/hVL mice or after immunization. The IgG concentrations were determined by ELISA.

DETAILED DESCRIPTION

The present disclosure relates to genetically modified animals and cells with humanized heavy chain immunoglobulin locus and/or humanized light chain immunoglobulin locus (e.g., kappa chain locus).

The genetically modified animals can be made by introducing human immunoglobulin genes into the genome of non-human animals to produce animals that can express humanized antibodies or chimeric antibodies. FIG. 1A shows the methods of making the humanized mice. In some embodiments, the methods first involve modifying the human immunoglobulin region on the human chromosome. The modified human chromosomes are then introduced into the mouse recipient cell. The human immunoglobulin variable region is then introduced into the corresponding region of the mouse genome by direct replacement (e.g., in one step replacement). The recipient cells are then screened, preferably for the cells that do not contain the human chromosomes. The cells are then injected to blastocysts to prepare chimeric animals (e.g., mice). Subsequent breeding can be performed to obtain animals containing intact humanized immunoglobulin locus.

The genetically modified animals described herein can have various advantages. For examples, in some cases, the genetically modified animals described herein have complete human antibody repertoires. Thus, the variable domains generated by these animals can have a diversity that is very similar to the diversity of the variable domains in human. Furthermore, because the entire sequence at the human immunoglobulin locus are introduced into the animal genome (with no modifications or limited modifications), these genes can undergo the V(D)J recombination in a way that is very similar to what happens in human. In addition, the antibody production can be very efficient and has a rate that is similar to the normal rates due to the efficient V(D)J recombination. In addition, because V(D)J recombination may occur between endogenous IGHV, IGHD, IGHJ, IGKV and IGKJ genes and human genes, if the endogenous IGHV, IGHD, IGHJ, IGKV and IGKJ genes are incorporated in the rearranged heavy chain VDJ segment or the rearranged light chain VJ segment, it is likely that the antibodies generated by the antibody repertoires have immunogenic epitopes in human. The immunogenicity can lead to production of anti-drug-antibodies and may comprise efficacy. Here, the endogenous IGHV, IGHD, IGHJ, IGKV and IGKJ genes have been effectively deleted. It is less likely that the antibodies generated by the antibody repertoires are immunogenic in humans. Thus, the antibodies are more suitable for being used as therapeutics in humans. Therefore, the genetically modified animals provide an advantageous platform to produce humanized antibodies.

As used herein, the term “antibody” refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable (VH) domain and a heavy chain constant region (CH). Each light chain comprises a light chain variable (VL) domain and a light chain constant region (CL). The VH and VL domains can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL comprises three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (heavy chain CDRs may be abbreviated as HCDR1, HCDR2 and HCDR3; light chain CDRs may be abbreviated as LCDR1, LCDR2 and LCDR3). The term “high affinity” antibody refers to an antibody that has a K_(D) with respect to its target epitope about of 10⁻⁹ M or lower (e.g., about or lower than 1×10⁻⁹ M, 1×10⁻¹⁰ M, 1×10⁻¹¹ M, or 1×10⁻¹² M). In some embodiments, K_(D) can be measured by surface plasmon resonance, e.g., BIACORE™ or ELISA.

As used herein, the term “antigen-binding fragment” refers to a portion of a full-length antibody, wherein the portion of the antibody is capable of specifically binding to an antigen. In some embodiments, the antigen-binding fragment contains at least one variable domain (e.g., a variable domain of a heavy chain or a variable domain of light chain). Non-limiting examples of antibody fragments include, e.g., Fab, Fab′, F(ab′)2, and Fv fragments.

As used herein, the term “human antibody” refers to an antibody that is encoded by a nucleic acid (e.g., rearranged human immunoglobulin heavy or light chain locus) present in a human. In some embodiments, a human antibody is collected from a human or produced in a human cell culture (e.g., human hybridoma cells). In some embodiments, a human antibody is produced in a non-human cell (e.g., a mouse or hamster cell line). In some embodiments, a human antibody is produced in a bacterial or yeast cell. In some embodiments, a human antibody is produced in a transgenic non-human animal (e.g., a mouse) containing an unrearranged or rearranged human immunoglobulin locus (e.g., heavy or light chain human immunoglobulin locus).

As used herein, the term “chimeric antibody” refers to an antibody that contains a sequence present in at least two different antibodies (e.g., antibodies from two different mammalian species such as a human and a mouse antibody). A non-limiting example of a chimeric antibody is an antibody containing the variable domain sequences (e.g., all or part of a light chain and/or heavy chain variable domain sequence) of a human antibody and the constant domains of a non-human antibody. Additional examples of chimeric antibodies are described herein and are known in the art.

As used herein, the term “humanized antibody” refers to a non-human antibody which contains sequence derived from a non-human (e.g., mouse) immunoglobulin and contains sequences derived from a human immunoglobulin.

As used herein, the term “single-chain antibody” refers to a single polypeptide that contains at least two immunoglobulin variable domains (e.g., a variable domain of a mammalian immunoglobulin heavy chain or light chain) that is capable of specifically binding to an antigen.

As used herein, the terms “subject” and “patient” are used interchangeably throughout the specification and describe an animal, human or non-human. Veterinary and non-veterinary applications are contemplated by the present disclosure. Human patients can be adult humans or juvenile humans (e.g., humans below the age of 18 years old). In addition to humans, patients include but are not limited to mice, rats, hamsters, guinea-pigs, rabbits, ferrets, cats, dogs, and primates. Included are, for example, non-human primates (e.g., monkey, chimpanzee, gorilla, and the like), rodents (e.g., rats, mice, gerbils, hamsters, ferrets, rabbits), lagomorphs, swine (e.g., pig, miniature pig), equine, canine, feline, bovine, and other domestic, farm, and zoo animals.

As used herein, when referring to an antibody, the phrases “specifically binding” and “specifically binds” mean that the antibody interacts with its target molecule preferably to other molecules, because the interaction is dependent upon the presence of a particular structure (i.e., the antigenic determinant or epitope) on the target molecule; in other words, the reagent is recognizing and binding to molecules that include a specific structure rather than to all molecules in general. An antibody that specifically binds to the target molecule may be referred to as a target-specific antibody.

As used herein, the terms “polypeptide,” “peptide,” and “protein” are used interchangeably to refer to polymers of amino acids of any length of at least two amino acids.

As used herein, the terms “polynucleotide,” “nucleic acid molecule,” and “nucleic acid sequence” are used interchangeably herein to refer to polymers of nucleotides of any length of at least two nucleotides, and include, without limitation, DNA, RNA, DNA/RNA hybrids, and modifications thereof.

As used herein, the term “an unmodified human sequence” refers to a sequence that is derived from a human subject, a human cell, a cultured human cell or a human cell line, wherein the sequence is identical to the genetic sequence of a human subject, a human cell, a cultured human cell or a human cell line.

Genetically Modified Heavy Chain Immunoglobulin Locus

Heavy chain immunoglobulin locus (also known as IGH or immunoglobulin heavy locus) is a region on the chromosome (e.g., human chromosome 14) that contains genes for the heavy chains of human antibodies (or immunoglobulins).

This region represents the germline organization of the heavy chain locus. The locus includes V (variable), D (diversity), J (joining), and C (constant) segments. The genes in the V region form a V gene cluster (also known as IGHV gene cluster). The genes in the D region form a D gene cluster (also known as IGHD gene cluster). The genes in the J region form a J gene cluster (also known as IGHJ gene cluster).

During B cell development, a recombination event at the DNA level joins a single D segment (also known as an IGHD gene) with a J segment (also known as an IGHJ gene); the fused D-J exon of this partially rearranged D-J region is then joined to a V segment (also known as an IGHV gene). The rearranged V-D-J region containing a fused V-D-J exon is then transcribed and fused at the RNA level to the IGHM constant region; this transcript encodes a mu heavy chain. Later in development B cells generate V-D-J-Cmu-Cdelta pre-messenger RNA, which is alternatively spliced to encode either a mu or a delta heavy chain. Mature B cells in the lymph nodes undergo switch recombination, so that the fused V-D-J gene segment is brought in proximity to one of the IGHG, IGHA, or IGHE gene segments and each cell expresses either the gamma, alpha, or epsilon heavy chain. Potential recombination of many different IGH genes with several IGHJ genes provides a wide range of antigen recognition. Additional diversity is attained by junctional diversity, resulting from the random addition of nucleotides by terminal deoxynucleotidyl transferase, and by somatic hypermutation, which occurs during B cell maturation in the spleen and lymph nodes. Several V, D, J, and C segments are known to be incapable of encoding a protein and are considered pseudogenous gene segments (often simply referred to as pseudogenes).

The human heavy chain immunoglobulin locus is located on human chromosome 14. Table 1 lists IGH genes and its relative orders in this locus.

TABLE 1 List of IGHV genes on human chromosome 14 Gene names Order IGHV(III)-82 1 IGHV7-81 2 IGHV4-80 3 IGHV3-79 4 IGHV(II)-78-1 5 IGHV5-78 6 IGHV7-77 7 IGHV(III)-76-1 8 IGHV3-76 9 IGHV3-75 10 IGHV(II)-74-1 11 IGHV3-74 12 IGHV3-73 13 IGHV3-72 14 IGHV3-71 15 IGHV2-70 16 IGHV1-69D 17 IGHV1-69-2 18 IGHV3-69-1 19 IGHV2-70D 20 IGHV1-69 21 IGHV1-68 22 IGHV(III)-67-4 23 IGHV(III)-67-3 24 IGHV(III)-67-2 25 IGHV(II)-67-1 26 SLC20A1P1 (GLVR1) 27 IGHV1-67 28 IGHV3-66 29 IGHV(II)-65-1 30 IGHV3-65 31 IGHV3-64 32 GOLGA4P3 (Golgin) 33 IGHV3-63 34 IGHV(II)-62-1 35 IGHV3-62 36 IGHV4-61 37 IGHV(II)-60-1 38 IGHV3-60 39 IGHV4-59 40 IGHV1-58 41 IGHV3-57 42 IGHV7-56 43 IGHV4-55 44 IGHV3-54 45 IGHV(II)-53-1 46 IGHV3-53 47 IGHV3-52 48 IGHV(II)-51-2 49 IGHV(III)-51-1 50 IGHV5-51 51 IGHV3-50 52 IGHV(II)-49-1 53 IGHV3-49 54 IGHV3-48 55 IGHV(III)-47-1 56 IGHV3-47 57 IGHV(II)-46-1 58 IGHV1-46 59 IGHV1-45 60 IGHV(II)-44-2 61 IGHV(IV)-44-1 62 IGHV(III)-44 63 IGHV(II)-43-1 64 IGHV3-43 65 IGHV3-42 66 IGHV3-41 67 IGHV(II)-40-1 68 IGHV7-40 69 IGHV4-39 70 IGHV1-38-4 71 IGHV(III)-38-1D 72 IGHV3-38-3 73 IGHV(III)-44D 74 IGHV(II)-43-1D 75 IGHV3-43D 76 IGHV3-42D 77 IGHV7-40D 78 IGHV4-38-2 79 IGHV(III)-38-1 80 IGHV3-38 81 IGHV3-37 82 IGHV3-36 83 IGHV3-35 84 IGHV7-34-1 85 IGHV4-34 86 IGHV3-33-2 87 IGHV(II)-33-1 88 IGHV3-33 89 GOLGA4P1 (Golgin) 90 IGHV3-32 91 IGHV(II)-31-1 92 IGHV4-31 93 IGHV3-30-52 94 IGHV(II)-30-51 95 IGHV3-30-5 96 IGHV3-30-42 97 IGHV(II)-30-41 98 IGHV4-30-4 99 IGHV3-30-33 100 IGHV(II)-30-32 101 IGHV3-30-3 102 IGHV3-30-22 103 IGHV(II)-30-21 104 IGHV4-30-2 105 IGHV4-30-1 106 IGHV3-30-2 107 IGHV(II)-30-1 108 IGHV3-30 109 GOLGA4P2 (Golgin) 110 IGHV3-29 111 IGHV(II)-28-1 112 IGHV4-28 113 IGHV7-27 114 IGHV(II)-26-2 115 IGHV(III)-26-1 116 IGHV2-26 117 IGHV(III)-25-1 118 IGHV3-25 119 IGHV1-24 120 IGHV3-23D 121 IGHV(III)-22-2D 122 IGHV(II)-22-1D 123 IGHV3-23 124 IGHV(III)-22-2 125 IGHV(II)-22-1 126 IGHV3-22 127 IGHV3-21 128 IGHV(II)-20-1 129 IGHV3-20 130 IGHV3-19 131 IGHV1-18 132 SLC20A1P2 133 IGHV1-17 134 IGHV(III)-16-1 135 IGHV3-16 136 IGHV(II)-15-1 137 IGHV3-15 138 IGHV1-14 139 IGHV(III)-13-1 140 IGHV3-13 141 IGHV1-12 142 IGHV(III)-11-1 143 IGHV3-11 144 IGHV2-10 145 IGHV3-9 146 IGHV1-8 147 IGHV5-10-1 148 IGHV3-64D 149 IGHV3-7 150 IGHV3-6 151 IGHV(III)-5-2 152 IGHV(III)-5-1 153 IGHV2-5 154 IGHV7-4-1 155 IGHV4-4 156 IGHV1-3 157 IGHV(III)-2-1 158 IGHV1-2 159 * * IGHV(II)-1-1 162 IGHV6-1 163 *

RPS8P1, ADAM6, and KIAA0125 are also located in this locus. The relative order of RPS8P1 is 160, the relative order of ADAM6 is161, and the relative order of KIAA0125 is 164. Table 2 lists all IGHD genes and its relative orders on human chromosome 14. Table 3 lists all IGHJ genes and its relative orders on human chromosome 14. The genes for immunoglobulin constant domains are located after the IGHV, IGHD, and IGHJ genes. These genes include (as shown in the following order): immunoglobulin heavy constant mu (IGHM), immunoglobulin heavy constant delta (IGH δ), immunoglobulin heavy constant gamma 3 (IGHG3), immunoglobulin heavy constant gamma 1 (IGHG1), immunoglobulin heavy constant epsilon P1 (pseudogene) (IGHEP1), immunoglobulin heavy constant alpha 1 (IGHA1), immunoglobulin heavy constant gamma P (non-functional) (IGHGP), immunoglobulin heavy constant gamma 2 (IGHG2), immunoglobulin heavy constant gamma 4 (IGHG4), immunoglobulin heavy constant epsilon (IGHE), and immunoglobulin heavy constant alpha 2 (IGHA2). These genes and the order of these genes are also shown in FIG. 37 and FIG. 41 .

TABLE 2 List of IGHD genes on human chromosome 14 Gene names Order IGHD1-1 165 IGHD2-2 166 IGHD3-3 167 IGHD4-4 168 IGHD5-5 169 IGHD6-6 170 IGHD1-7 171 IGHD2-8 172 IGHD3-9 173 IGHD3-10 174 IGHD4-11 175 IGHD5-12 176 IGHD6-13 177 IGHD1-14 178 IGHD2-15 179 IGHD3-16 180 IGHD4-17 181 IGHD5-18 182 IGHD6-19 183 IGHD1-20 184 IGHD2-21 185 IGHD3-22 186 IGHD4-23 187 IGHD5-24 188 IGHD6-25 189 IGHD1-26 190 * IGHD7-27 192

TABLE 3 List of IGHJ genes on human chromosome 14 Gene names Order IGHJ1P 191 IGHJ1 193 IGHJ2 194 IGHJ2P 195 IGHJ3 196 IGHJ4 197 IGHJ5 198 IGHJ3P 199 IGHJ6 200

The mouse heavy chain immunoglobulin locus is located on mouse chromosome 12. Table 4 lists IGHV genes and its relative orders in this locus.

TABLE 4 List of IGHV genes on mouse chromosome 12 Gene names Order IGHV1-86 1 IGHV1-85 2 IGHV1-84 3 IGHV1-83 4 IGHV1-82 5 IGHV1-81 6 IGHV1-80 7 IGHV1-79 8 IGHV1-78 9 IGHV1-77 10 IGHV8-16 11 IGHV1-76 12 IGHV8-15 13 IGHV1-75 14 IGHV8-14 15 IGHV1-74 16 IGHV1-73 17 IGHV8-13 18 IGHV1-72 19 IGHV1-71 20 IGHV1-70 21 IGHV8-12 22 IGHV1-69 23 IGHV1-68 24 IGHV1-67 25 IGHV1-66 26 IGHV8-11 27 IGHV1-65 28 IGHV8-10 29 IGHV1-64 30 IGHV1-63 31 IGHV8-9 32 IGHV1-62-3 33 IGHV1-62-2 34 IGHV1-62-1 35 IGHV1-62 36 IGHV1-61 37 IGHV1-60 38 IGHV1-59 39 IGHV1-58 40 IGHV8-8 41 IGHV1-57 42 IGHV8-7 43 IGHV1-56 44 IGHV1-55 45 IGHV1-54 46 IGHV8-6 47 IGHV1-53 48 IGHV1-52 49 IGHV1-51 50 IGHV1-50 51 IGHV8-5 52 IGHV1-49 53 IGHV1-48 54 IGHV8-4 55 IGHV8-3 56 IGHV1-47 57 IGHV1-46 58 IGHV1-45 59 IGHV1-44 60 IGHV1-43 61 IGHV1-42 62 IGHV1-41 63 IGHV1-40 64 IGHV1-39 65 IGHV1-38 66 IGHV1-37 67 IGHV1-36 68 IGHV1-35 69 IGHV1-34 70 IGHV1-33 71 IGHV1-32 72 IGHV1-31 73 IGHV1-30 74 IGHV1-29 75 IGHV1-28 76 IGHV1-27 77 IGHV1-26 78 IGHV1-25 79 IGHV1-24 80 IGHV1-23 81 IGHV1-22 82 IGHV1-21 83 IGHV1-21-1 84 IGHV1-20 85 IGHV1-19 86 IGHV1-19-1 87 IGHV1-18 88 IGHV1-17 89 IGHV1-17-1 90 IGHV1-16 91 IGHV1-15 92 IGHV1-14 93 IGHV1-13 94 IGHV1-12 95 IGHV1-11 96 IGHV1-10 97 IGHV1-9 98 IGHV15-2 99 IGHV1-8 100 IGHV10-4 101 IGHV1-7 102 IGHV1-6 103 IGHV10-3 104 IGHV1-5 105 IGHV10-2 106 IGHV1-4 107 IGHV1-3 108 IGHV10-1 109 IGHV1-2 110 IGHV8-2 111 IGHV6-7 112 IGHV6-6 113 IGHV6-5 114 IGHV6-4 115 IGHV6-3 116 IGHV12-3 117 IGHV13-2 118 IGHV1-1 119 IGHV8-1 120 IGHV3-8 121 IGHV5-21 122 IGHV3-7 123 IGHV9-4 124 IGHV3-6 125 IGHV13-1 126 IGHV3-5 127 IGHV3-4 128 IGHV7-4 129 IGHV3-3 130 IGHV14-4 131 IGHV15-1 132 IGHV7-3 133 IGHV9-3 134 IGHV12-2 135 IGHV9-2 136 IGHV12-1 137 IGHV9-1 138 IGHV6-2 139 IGHV16-1 140 IGHV14-3 141 IGHV11-2 142 IGHV3-2 143 IGHV4-2 144 IGHV14-2 145 IGHV11-1 146 IGHV3-1 147 IGHV4-1 148 IGHV14-1 149 IGHV7-2 150 IGHV7-1 151 IGHV5-19 152 IGHV2-9 153 IGHV2-8 154 IGHV5-18 155 IGHV5-17 156 IGHV5-16 157 IGHV5-15 158 IGHV2-7 159 IGHV2-6-8 160 IGHV2-9-1 161 IGHV5-12-4 162 IGHV5-9-1 163 IGHV2-6 164 IGHV5-12 165 IGHV5-11 166 IGHV2-5 167 IGHV5-10 168 IGHV5-9 169 IGHV5-8 170 IGHV2-4 171 IGHV5-7 172 IGHV5-6 173 IGHV5-5 174 IGHV2-3 175 IGHV6-1 176 IGHV5-4 177 IGHV5-3 178 IGHV2-2 179 IGHV5-2 180 IGHV2-1 181 IGHV5-1 182

Table 5 lists all IGHD genes and its relative orders on mouse chromosome 12. Table 6 lists all IGHJ genes and its relative orders on mouse chromosome 12. The genes for immunoglobulin constant domains are after the IGHV, IGHD, and IGHJ genes. These genes include (as shown in the following order): immunoglobulin heavy constant mu (IGHM), immunoglobulin heavy constant delta (IGH δ), immunoglobulin heavy constant gamma 3 (IGHG3), immunoglobulin heavy constant gamma 1 (IGHG1), immunoglobulin heavy constant gamma 2b (IGHG2b), immunoglobulin heavy constant gamma 2a (IGHG2a), immunoglobulin heavy constant epsilon (IGHE), and immunoglobulin heavy constant alpha (IGHA) genes. These genes and the order of these genes are also shown in FIG. 38 and FIG. 42 .

TABLE 5 List of IGHD genes on mouse chromosome 12 Gene names Order IGHD5-1 183 IGHD3-1 184 IGHD1-1 185 IGHD6-1 186 IGHD2-3 187 IGHD6-2 188 IGHD2-4 189 IGHD2-5 191 IGHD5-3 192 IGHD5-7 193 IGHD2-6 194 IGHD5-4 195 IGHD5-8 196 IGHD2-7 197 IGHD5-5 198 IGHD2-8 199 IGHD5-6 200 IGHD3-2 201 IGHD4-1 202

TABLE 6 List of IGHJ genes on mouse chromosome 12 Gene names Order IGHJ1 203 IGHJ2 204 IGHJ3 205 IGHJ4 206

The present disclosure provides genetically-modified, non-human animal comprising one or more human IGHV genes, one or more human IGHD genes, and/or one or more human IGHJ genes. In some embodiments, the human IGHV genes, the human IGHD genes, and the human IGHJ genes are operably linked together and can undergo VDJ rearrangement. In some embodiments, the human IGHV genes, the human IGHD genes, and the human IGHJ genes are at the endogenous heavy chain immunoglobulin gene locus.

In some embodiments, the animal compromises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160 or 161 human IGHV genes (e.g., genes as shown in Table 1).

In some embodiments, the animal compromises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76, and IGHV3-75.

In some embodiments, the animal compromises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGHV(III)-5-2, IGHV(III)-5-1, IGHV2-5, IGHV7-4-1, IGHV4-4, IGHV1-3, IGHV(III)-2-1, IGHV1-2, IGHV(II)-1-1, and IGHV6-1.

In some embodiments, the animal compromises an unmodified human sequence comprising a sequence starting from a gene selected from IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76, and IGHV3-75, and ending at a gene selected from IGHV(III)-5-2, IGHV(III)-5-1, IGHV2-5, IGHV7-4-1, IGHV4-4, IGHV1-3, IGHV(III)-2-1, IGHV1-2, IGHV(II)-1-1, and IGHV6-1. In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHV1-2. In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHV(II)-1-1. In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHV-6-1.

In some embodiments, the animal compromises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 human IGHD genes (e.g., genes as shown in Table 2). In some embodiments, the animal compromises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGHD1-1, IGHD2-2, IGHD3-3, IGHD4-4, IGHD5-5, IGHD4-23, IGHD5-24, IGHD6-25, IGHD1-26, and IGHD7-27.

In some embodiments, the animal compromises about or at least 1, 2, 3, 4, 5, 6, 7, 8, or 9 human IGHJ genes (e.g., genes as shown in Table 3). In some embodiments, the animal compromises 1, 2, 3, 4, 5, 6, 7, 8, or 9 human IGHJ genes selected from IGHJ1P, IGHJ1, IGHJ2, IGHJ2P, IGHJ3, IGHJ4, IGHJ5, IGHJ3P, and IGHJ6.

In some embodiments, the animal compromises an unmodified human sequence comprising a sequence starting from a gene selected from IGHD1-1, IGHD2-2, IGHD3-3, IGHD4-4, IGHD5-5, IGHD4-23, IGHD5-24, IGHD6-25, IGHD1-26, and IGHD7-27, and ending at a gene selected from IGHJ1P, IGHJ1, IGHJ2, IGHJ2P, IGHJ3, IGHJ4, IGHJ5, IGHJ3P, and IGHJ6. In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHD1-1 to human IGHJ6.

In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHD1-1 to human IGHD7-27.

In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHJ1P to human IGHJ6.

In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHJ1 to human IGHJ6.

In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(III)-82 to human IGHJ6.

In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV1-2 to human IGHJ6.

In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV(II)-1-1 to human IGHJ6. In some embodiments, the unmodified human sequence derived from a human heavy chain immunoglobulin gene locus starting from human IGHV6-1 to human IGHJ6.

In some embodiments, the animal can have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences. In some embodiments, the unmodified human sequence has a length of about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 kb.

In some embodiments, the animal comprises one or more endogenous genes selected from the group consisting of immunoglobulin heavy constant mu (IGHM), immunoglobulin heavy constant delta (IGHδ), immunoglobulin heavy constant gamma 3 (IGHG3), immunoglobulin heavy constant gamma 1 (IGHG1), immunoglobulin heavy constant gamma 2b (IGHG2b), immunoglobulin heavy constant gamma 2a (IGHG2a), immunoglobulin heavy constant epsilon (IGHE), and immunoglobulin heavy constant alpha (IGHA) genes. In some embodiments, these endogenous genes are operably linked together. In some embodiments, these endogenous genes have the same order as in a wildtype animal. In some embodiments, isotype switching (immunoglobulin class switching) can occur in the animal.

In some embodiments, the IGHV genes, the IGHD genes, and/or the IGHJ genes are operably linked together. The VDJ recombination can occur among these genes and produce functional antibodies. In some embodiments, these genes are arranged in an order that is similar to the order in human heavy chain immunoglobulin locus. This arrangement offers various advantages, e.g., the arrangement of these genes allow the production of heavy chain variable domains with a diversity that is very similar to the diversity of the heavy chain variable domains in human. As some random sequences may be inserted to the sequence during VDJ recombination, in some embodiments, the complete human antibody repertoires with no or minimum modifications can reduce the likelihood that non-human sequence is inserted during the VDJ recombination.

In some embodiments, the IGHV genes, the IGHD genes, and/or the IGHJ genes are operably linked together to one or more genes (e.g., all genes) selected from IGHM, IGHδ, IGHG3, IGHG1, IGHG2b, IGHG2a, IGHE, and IGHA genes.

In some embodiments, the animal comprises a disruption in the animal's endogenous heavy chain immunoglobulin gene locus. In some embodiments, the disruption in the animal's endogenous heavy chain immunoglobulin gene locus comprises a deletion of one or more endogenous IGHV genes, one or more endogenous IGHD genes, and one or more endogenous IGHJ genes.

In some embodiments, the animal is a mouse. The disruption in the animal's endogenous heavy chain immunoglobulin gene locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, or 182 mouse IGHV genes (e.g., genes as shown in Table 4). In some embodiments, the disruption compromises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV1-86, IGHV1-85, IGHV1-84, IGHV1-83, IGHV1-82, IGHV1-81, IGHV1-80, IGHV1-79, IGHV1-78, and IGHV1-77. In some embodiments, the mouse still compromises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV1-86, IGHV1-85, IGHV1-84, IGHV1-83, IGHV1-82, IGHV1-81, IGHV1-80, IGHV1-79, IGHV1-78, and IGHV1-77 (e.g., IGHV1-86).

In some embodiments, the disruption compromises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV5-6, IGHV5-5, IGHV2-3, IGHV6-1, IGHV5-4, IGHV5-3, IGHV2-2, IGHV5-2, IGHV2-1, and IGHV5-1. In some embodiments, the mouse still compromises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHV genes selected from IGHV5-6, IGHV5-5, IGHV2-3, IGHV6-1, IGHV5-4, IGHV5-3, IGHV2-2, IGHV5-2, IGHV2-1, and IGHV5-1.

In some embodiments, the disruption in the animal's endogenous heavy chain immunoglobulin gene locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 mouse IGHD genes (e.g., genes as shown in Table 5). In some embodiments, the disruption compromises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2, and IGHD4-1. In some embodiments, the mouse still compromises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGHD genes selected from IGHD5-1, IGHD3-1, IGHD1-1, IGHD6-1, IGHD2-3, IGHD2-7, IGHD2-8, IGHD5-6, IGHD3-2, and IGHD4-1.

In some embodiments, the disruption compromises a deletion of about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4. In some embodiments, the mouse still compromises about or at least 1, 2, 3, or 4 mouse IGHJ genes selected from IGHJ1, IGHJ2, IGHJ3, and IGHJ4.

In some embodiments, the disruption in the animal's endogenous heavy chain immunoglobulin gene locus comprises a deletion of about or at least 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, or 3000 kb of an endogenous sequence.

In some embodiments, the deleted sequence starts from IGHV1-86 to IGHJ4, from IGHV1-85 to IGHJ4, from IGHV1-84 to IGHJ4, from IGHV1-83 to IGHJ4, or from IGHV1-82 to IGHJ4 (e.g., from IGHV1-85 to IGHJ4).

In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence in the human heavy chain immunoglobulin gene locus. In some embodiments, the sequence has a length of about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 or 3500 kb. In some embodiments, the sequence starts from human IGHV(III)-82 to IGHV1-2. In some embodiments, the sequence starts from human IGHV7-81 to IGHV1-2. In some embodiments, the sequence starts from human IGHV(II)-1-1 to IGHVJ6. In some embodiments, the sequence starts from human IGHV6-1 to IGHVJ6.

The human IGHV genes, the human IGHD genes, and the human IGHJ genes are operably linked together and can undergo VDJ rearrangement. In some embodiments, the modified mouse has complete human IGHV, IGHD, and IGHJ gene repertoires (e.g., including all non-pseudo human IGHV, IGHD, and IGHJ genes). Thus, the modified mouse can produce a complete human antibody repertory. In some embodiments, after VDJ recombination, one IGHV gene (e.g., IGHV3-21 or IGHV3-74) in Table 15 contributes to the sequence that encodes an antibody heavy chain variable region. One IGHD gene in Table 15 contributes to the sequence that encodes an antibody heavy chain variable region. And one IGHJ gene in Table 15 contributes to the sequence that encodes an antibody heavy chain variable region. In some embodiments, the IGHV gene is IGHV3-21 or IGHV3-74.

In some embodiments, one IGHV gene (e.g., IGHV3-30, IGHV3-33, IGHV4-39, or IGHV4-34) in FIG. 70A and FIG. 70B contributes to the sequence that encodes an antibody heavy chain variable region. One IGHD gene (e.g., IGHD6-19) in FIG. 70C contributes to the sequence that encodes an antibody heavy chain variable region. And one IGHJ gene (e.g., IGHJ4 or IGHJ6) in FIG. 70D contributes to the sequence that encodes an antibody heavy chain variable region. In some embodiments, one IGKV gene (e.g., IGKV4-1, IGKV1-33, IGKV2-30) in FIG. 71A and FIG. 71B contributes to the sequence that encodes an antibody light chain variable region. One IGKJ gene (e.g., IGKJ1, IGKJ2, or IGKJ4) in FIG. 71C contributes to the sequence that encodes an antibody light chain variable region.

Furthermore, in some cases, the entire mouse IGHV genes, IGHD genes, and IGHJ genes (e.g., including all none-pseudo genes) are knocked out, and the heavy chain variable region will not have any sequence that is encoded by a sequence derived from the mouse, thereby minimizing immunogenicity in human.

Genetically Modified Kappa Light Chain Immunoglobulin Locus

Kappa chain immunoglobulin locus (also known as IGK or immunoglobulin kappa locus) is a region on the chromosome (e.g., human chromosome 2) that contains genes for the light chains of human antibodies (or immunoglobulins). Similarly, the immunoglobulin light chain genes can also undergo a series rearrangement that lead to the production of a mature immunoglobulin light-chain nucleic acid (e.g., a kappa chain).

The joining of a V segment (also known as an IGKV gene) and a J segment (also known as an IGKJ gene) creates a continuous exon that encodes the whole of the light-chain variable domain. In the unrearranged DNA, the V gene segments (or IGKV gene cluster) are located relatively far away from the C region. The J gene segments (or IGKJ gene cluster) are located close to the C region. Joining of a V segment to a J gene segment also brings the V gene close to a C-region sequence. The J gene segment of the rearranged V region is separated from a C-region sequence only by an intron. To make a complete immunoglobulin light-chain messenger RNA, the V-region exon is joined to the C-region sequence by RNA splicing after transcription.

The human light chain immunoglobulin locus is located on human chromosome 2. Table 7 lists IGKV genes and its relative orders in this locus. There are several different groups for human IGKV genes, including IGKV1 genes (including all IGKV genes starting with IGKV1, also known as VκI), IGKV2 genes (including all IGKV genes starting with IGKV2, also known as VκII), IGKV3 genes (including all IGKV genes starting with IGKV3, also known as VκIII), IGKV4 genes (including all IGKV genes starting with IGKV4, also known as VκIV), IGKV5 genes (including all IGKV genes starting with IGKV5, also known as VκV), IGKV6 genes (including all IGKV genes starting with IGKV6, also known as VκVI), and IGKV7 genes (including all IGKV genes starting with IGKV7, also known as VκVII).

These IGKV genes in human chromosome 2 also form two clusters, the proximal Vκ cluster and the distal Vκ cluster (FIG. 28 ). The sequences in the two clusters are similar but are not identical. This large segmental duplication of the sequence occurred since the divergence of the human lineage from the most recent shared ancestor with other great apes. The relevant IGVK genes in each cluster is summarized in FIG. 64 .

TABLE 7 List of IGKV genes on human chromosome 2 Gene names Order IGKV3D-7 1 IGKV1D-8 2 IGKV1D-43 3 IGKV1D-42 4 IGKV2D-10 5 IGKV3D-11 6 IGKV1D-12 7 IGKV1D-13 8 IGKV2D-14 9 IGKV3D-15 10 IGKV1D-16 11 IGKV1D-17 12 IGKV6D-41 13 IGKV2D-18 14 IGKV2D-19 15 IGKV3D-20 16 IGKV6D-21 17 IGKV1D-22 18 IGKV2D-23 19 IGKV2D-24 20 IGKV3D-25 21 IGKV2D-26 22 IGKV1D-27 23 IGKV2D-28 24 IGKV2D-29 25 IGKV2D-30 26 IGKV3D-31 27 IGKV1D-32 28 IGKV1D-33 29 IGKV3D-34 30 IGKV1D-35 31 IGKV2D-36 32 IGKV1D-37 33 IGKV2D-38 34 IGKV1D-39 35 IGKV2D-40 36 IGKV2-40 37 IGKV1-39 38 IGKV2-38 39 IGKV1-37 40 IGKV2-36 41 IGKV1-35 42 IGKV3-34 43 IGKV1-33 44 IGKV1-32 45 IGKV3-31 46 IGKV2-30 47 IGKV2-29 48 IGKV2-28 49 IGKV1-27 50 IGKV2-26 51 IGKV3-25 52 IGKV2-24 53 IGKV2-23 54 IGKV1-22 55 IGKV6-21 56 IGKV3-20 57 IGKV2-19 58 IGKV2-18 59 IGKV1-17 60 IGKV1-16 61 IGKV3-15 62 IGKV2-14 63 IGKV1-13 64 IGKV1-12 65 IGKV3-11 66 IGKV2-10 67 IGKV1-9 68 IGKV1-8 69 IGKV3-7 70 IGKV1-6 71 IGKV1-5 72 IGKV2-4 73 IGKV7-3 74 IGKV5-2 75 IGKV4-1 76

Table 8 lists all IGKJ genes and its relative orders on human chromosome 2. The immunoglobulin kappa constant (IGKC) gene, which encodes the light chain immunoglobulin constant domains is located after the IGKV and IGKJ genes. These genes and the order of these genes are also shown in FIG. 39 and FIG. 43 .

TABLE 8 List of IGKJ genes on human chromosome 2 Gene names Order IGKJ1 77 IGKJ2 78 IGKJ3 79 IGKJ4 80 IGKJ5 81

The mouse light chain immunoglobulin locus is located on mouse chromosome 6. Table 9 lists IGKV genes and its relative orders in this locus.

TABLE 9 List of IGKV genes on mouse chromosome 6 Gene names Order IGKV2-137 1 IGKV1-136 2 IGKV1-135 3 * IGKV14-134-1 5 IGKV17-134 6 IGKV1-133 7 IGKV1-132 8 IGKV1-131 9 IGKV14-130 10 IGKV9-129 11 IGKV9-128 12 IGKV17-127 13 IGKV14-126-1 14 IGKV14-126 15 IGKV11-125 16 IGKV9-124 17 IGKV9-123 18 IGKV1-122 19 IGKV17-121 20 IGKV9-120 21 IGKV9-119 22 IGKV14-118-2 23 IGKV14-118-1 24 IGKV11-118 25 IGKV1-117 26 IGKV2-116 27 IGKV1-115 28 IGKV11-114 29 IGKV2-113 30 IGKV2-112 31 IGKV14-111 32 IGKV1-110 33 IGKV2-109 34 IGKV1-108 35 IGKV2-107 36 IGKV11-106 37 IGKV2-105 38 IGKV16-104 39 IGKV15-103 40 IGKV15-102 41 IGKV20-101-2 42 IGKV15-101-1 43 IGKV15-101 44 IGKV 14-100 45 IGKV1-99 46 IGKV12-98 47 IGKV15-97 48 IGKV10-96 49 IGKV2-95-2 50 IGKV2-95-1 51 IGKV10-95 52 IGKV10-94 53 IGKV2-93-1 54 IGKV19-93 55 IGKV4-92 56 IGKV4-91 57 IGKV4-90 58 IGKV13-89-1 59 IGKV12-89 60 IGKV1-88 61 IGKV13-87 62 IGKV4-86 63 IGKV13-85 64 IGKV13-84 65 IGKV4-83 66 IGKV13-82 67 IGKV4-81 68 IGKV13-80-1 69 IGKV4-80 70 IGKV4-79 71 IGKV13-78-1 72 IGKV4-78 73 IGKV4-77 74 IGKV13-76 75 IGKV4-75 76 IGKV13-74-1 77 IGKV4-74 78 IGKV13-73-1 79 IGKV4-73 80 IGKV4-72 81 IGKV13-71-1 82 IGKV4-71 83 IGKV4-70 84 IGKV4-69 85 IGKV4-68 86 IGKV12-67 87 IGKV12-66 88 IGKV4-65 89 IGKV13-64 90 IGKV4-63 91 IGKV13-62-1 92 IGKV4-62 93 IGKV13-61-1 94 IGKV4-61 95 IGKV4-59 96 IGKV4-60 97 IGKV4-58 98 IGKV13-57-2 99 IGKV4-57-1 100 IGKV13-57-1 101 IGKV4-57 102 IGKV13-56-1 103 IGKV4-56 104 IGKV13-55-1 105 IGKV4-55 106 IGKV13-54-1 107 IGKV4-54 108 IGKV4-53 109 IGKV4-52 110 IGKV4-51 111 IGKV4-50 112 IGKV12-49 113 IGKV5-48 114 IGKV12-47 115 IGKV12-46 116 IGKV5-45 117 IGKV12-44 118 IGKV5-43 119 IGKV12-42 120 IGKV12-41 121 IGKV5-40-1 122 IGKV12-40 123 IGKV5-39 124 IGKV12-38 125 IGKV5-37 126 IGKV18-36 127 IGKV1-35 128 IGKV8-34 129 IGKV7-33 130 IGKV6-32 131 IGKV8-31 132 IGKV8-30 133 * IGKV6-29 135 IGKV8-28 136 IGKV8-27 137 IGKV8-26 138 IGKV6-25 139 IGKV8-24 140 IGKV8-23-1 141 IGKV6-23 142 IGKV8-22 143 IGKV8-21 144 IGKV6-20 145 IGKV8-19 146 IGKV8-18 147 IGKV6-17 148 IGKV8-16 149 IGKV6-15 150 IGKV6-14 151 IGKV6-13 152 IGKV3-12-1 153 IGKV3-12 154 IGKV3-11 155 IGKV3-10 156 IGKV3-9 157 IGKV3-8 158 IGKV3-7 159 IGKV3-6 160 IGKV3-5 161 IGKV3-4 162 IGKV3-3 163 IGKV3-2 164 IGKV3-1 165

Gm9728 and Amd-ps2 are also located in this locus. The relative order of Gm9728 is 4, and the relative order of Amd-ps2 is 134. Table 10 lists all IGKJ genes and its relative orders on mouse chromosome 6. The IGKC gene, which encodes the light chain immunoglobulin constant domains are after the IGKV and IGKJ genes. These genes and the order of these genes are also shown in FIG. 40 and FIG. 44 .

TABLE 10 List of IGKJ genes on mouse chromosome 6 Gene names Order IGKJ1 166 IGKJ2 167 IGKJ3 168 IGKJ4 169 IGKJ5 170

The present disclosure provides genetically-modified, non-human animal comprising one or more human IGKV genes and/or one or more human IGKJ genes. In some embodiments, the human IGKV genes and the human IGKJ genes are operably linked together and can undergo VJ rearrangement. In some embodiments, the human IGKV genes and the human IGKJ genes are at endogenous light chain immunoglobulin gene locus.

In some embodiments, the animal compromises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, or 76 human IGKV genes (e.g., genes as shown in Table 7).

In some embodiments, the animal compromises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGKV3D-7, IGKV1D-8, IGKV1D-43, IGKV1D-42, IGKV2D-10, IGKV3D-11, IGKV1D-12, IGKV1D-13, IGKV2D-14, and IGKV3D-15.

In some embodiments, the animal compromises 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 genes selected from IGKV2-10, IGKV1-9, IGKV1-8, IGKV3-7, IGKV1-6, IGKV1-5, IGKV2-4, IGKV7-3, IGKV5-2, and IGKV4-1.

In some embodiments, the animal compromises about or at least 1, 2, 3, 4, or 5 human IGKJ genes (e.g., genes as shown in Table 3). In some embodiments, the animal compromises 1, 2, 3, 4, or 5 human IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5.

In some embodiments, the animal comprises an endogenous IGKC. In some embodiments, the IGKV genes and/or the IGKJ genes are operably linked together. The VJ recombination can occur among these genes and produce functional antibodies. In some embodiments, these genes are arranged in an order that is similar to the order in human light chain immunoglobulin locus. This arrangement offers various advantages, e.g., the arrangement of these genes allow the production of light chain variable domains with a diversity that is very similar to the diversity of the light chain variable domains in human.

In some embodiments, the IGKV genes and/or the IGKJ genes are operably linked together to the IGKC gene (e.g., endogenous IGKC gene).

In some embodiments, the animal comprises a disruption in the animal's endogenous light chain immunoglobulin gene locus. In some embodiments, the disruption in the animal's endogenous light chain immunoglobulin gene locus comprises a deletion of one or more endogenous IGKV genes, and one or more endogenous IGKJ genes.

In some embodiments, the animal is a mouse. The disruption in the animal's endogenous heavy chain immunoglobulin gene locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160, 161, 162, or 163 mouse IGKV genes (e.g., genes as shown in Table 9). In some embodiments, the disruption compromises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV2-137, IGKV1-136, IGKV1-135, IGKV14-134-1, IGKV17-134, IGKV1-133, IGKV1-132, IGKV1-131, IGKV14-130, and IGKV9-129. In some embodiments, the mouse still compromises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV2-137, IGKV1-136, IGKV1-135, IGKV14-134-1, IGKV17-134, IGKV1-133, IGKV1-132, IGKV1-131, IGKV14-130, and IGKV9-129.

In some embodiments, the disruption compromises a deletion of about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV3-10, IGKV3-9, IGKV3-8, IGKV3-7, IGKV3-6, IGKV3-5, IGKV3-4, IGKV3-3, IGKV3-2, and IGKV3-1. In some embodiments, the mouse still compromises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mouse IGKV genes selected from IGKV3-10, IGKV3-9, IGKV3-8, IGKV3-7, IGKV3-6, IGKV3-5, IGKV3-4, IGKV3-3, IGKV3-2, and IGKV3-1.

In some embodiments, the disruption compromises a deletion of about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5. In some embodiments, the mouse still compromises about or at least 1, 2, 3, 4, or 5 mouse IGKJ genes selected from IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5 (e.g., IGKJ5).

In some embodiments, the disruption in the animal's endogenous kappa light chain immunoglobulin gene locus comprises a deletion of about or at least 500 kb, 600 kb, 700 kb, 800 kb, 900 kb, 1000 kb, 1500 kb, 2000 kb, 2500 kb, 3000 kb or 3500 kb of an endogenous sequence.

In some embodiments, the deleted sequence starts from IGKV2-137 to IGKJ4, from IGKV1-136 to IGKJ4, from IGKV1-135 to IGKJ4, from IGKV2-137 to IGKJ5, from IGKV1-136 to IGKJ5, or from IGKV1-135 to IGKJ5 (e.g., from IGKV2-137 to IGKJ5).

In some embodiments, the animal comprises about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 sequences that are at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a sequence in the human light chain immunoglobulin gene locus. In some embodiments, the sequence has a length of about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 or 3500 kb.

In some embodiments, the animal can have one, two, three, four, five, six, seven, eight, nine, or ten unmodified human sequences. In some embodiments, the unmodified human sequence has a length of about or at least 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000 or 3500 kb.

In some embodiments, the sequence starts from human IGKV3D-7 to IGKJ5. In some embodiments, the sequence starts from human IGKV3D-7 to IGKJ4. In some embodiments, the sequence starts from human IGKV1D-8 to IGKJ5. In some embodiments, the sequence starts from human IGKV1D-8 to IGKJ4.

The human IGKV genes and the human IGKJ genes are operably linked together and can undergo VJ rearrangement. In some embodiments, the modified mouse has complete human IGKV and IGKJ gene repertoires (e.g., including all non-pseudo human IGKV and IGKJ genes). Thus, the modified mouse can produce a complete human antibody repertory. In some embodiments, after VJ recombination, one IGKV gene (e.g., IGKV1D-43, IGKV1D-13, IGKV1D-16, or IGKV1D-12) in Table 16 contributes to the sequence that encodes an antibody light chain variable region. One human IGKJ gene contributes to the sequence that encodes an antibody light chain variable region. In some embodiments, the IGKV gene is IGKV1D-43, IGKV1D-13, IGKV1D-16, or IGKV1D-12. Furthermore, in some cases, the entire mouse IGKV genes, and IGKJ genes (all none-pseudo genes) are knocked out, and the light chain variable region will not have any sequence that is encoded by a sequence derived from the mouse, thereby minimizing immunogenicity in humans.

In some embodiments, the human proximal Vκ cluster IGKV genes are included in the modified chromosome. In some embodiments, the human distal Vκ cluster IGKV genes are included in the modified chromosome. In some embodiments, both the human proximal Vκ cluster IGKV genes and the human distal Vκ cluster IGKV genes are included in the modified chromosome.

Genetically Modified Lambda Light Chain Immunoglobulin Locus

Lambda chain immunoglobulin locus (also known as IGL or immunoglobulin lambda locus) is a region on the chromosome (e.g., human chromosome 22) that contains genes for the light chains of human antibodies (or immunoglobulins). Similarly, the immunoglobulin light chain genes can also undergo a series rearrangement that lead to the production of a mature immunoglobulin light-chain nucleic acid (e.g., a lambda chain). In a healthy human individual, the total kappa to lambda ratio is roughly 2:1 in serum (measuring intact whole antibodies) or 1:1.5 if measuring free light chains. In mice, the total kappa to lambda ratio is roughly 9:1.

In some embodiments, the animal comprises a human lambda chain immunoglobulin locus.

In some embodiments, the animal comprises a disruption in the animal's endogenous lambda light chain immunoglobulin gene locus. In some embodiments, the disruption in the animal's endogenous light chain immunoglobulin gene locus comprises a deletion of one or more endogenous IGLV genes, one or more endogenous IGLJ genes, and/or one or more immunoglobulin lambda constant (IGLC) genes (e.g., IGLC1, IGLC2, IGLC3, and IGLC4).

The mouse lambda light chain immunoglobulin locus (IGL locus) is located on mouse chromosome 16. Table 11 lists IGLV, IGLJ, and IGLC genes and its relative orders in this locus.

TABLE 11 List of genes at mouse IGL locus Gene Reference IMGT Chromosomal orientation on NCBI GRCm38.p3 Gene positions Gene localization chromosome Gene ID C57BL/6J in sequence IGLV2 16A3 (11.93 cM) REV 110612 NC_000082.6 19260403 . . . 19260844 IGLV3 16A3 (11.91 cM) REV 404743 NC_000082.6 19241208 . . . 19241679 IGLJ2 16A3 (11.89 cM) REV 404739 NC_000082.6 19200198 . . . 19200235 IGLC2 16A3 (11.89 cM) REV 110786 NC_000082.6 19198536 . . . 19198852 IGLJ4 16A3 (11.89 cM) REV 404742 NC_000082.6 19196495 . . . 19196536 IGLC4 16A3 (11.89 cM) REV 404736 NC_000082.6 19194999 . . . 19195312 IGLV1 16A3 (11.82 cM) REV 16142 NC_000082.6 19085017 . . . 19085460 IGLJ3 16A3 (11.81 cM) REV 404740 NC_000082.6 19067041 . . . 19067078 IGLJ3P 16A3 (11.81 cM) REV 404741 NC_000082.6 19066371 . . . 19066408 IGLC3 16A3 (11.81 cM) REV 110787 NC_000082.6 19065365 . . . 19065681 IGLJ1 16A3 (11.81 cM) REV 404737 NC_000082.6 19063225 . . . 19063262 IGLC1 16A3 (11.80 cM) REV 110785 NC_000082.6 19061752 . . . 19062071

The disruption in the animal's endogenous lambda light chain immunoglobulin gene locus comprises a deletion of at least or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 mouse IGLV, IGLJ, and IGLC genes (e.g., genes as shown in Table 11). In some embodiments, the deletion compromises about or at least 1, 2, 3, or 4 mouse IGKC genes selected from IGLC1, IGLC2, IGLC3, and IGLC4. In some embodiments, the disruption compromises a deletion of about or at least 1, 2, or 3 mouse IGLV genes selected from IGLV1, IGLV2, and IGLV3. In some embodiments, the disruption compromises a deletion of about or at least 1, 2, 3, 4, or 5 mouse IGLJ genes selected from IGLJ1, IGLJ2, IGLJ3, IGLJ3P, and IGLJ4.

In some embodiments, the disruption in the animal's endogenous lambda light chain immunoglobulin gene locus comprises a deletion of about or at least 10 kb, 20 kb, 30 kb, 40 kb, 50 kb, 60 kb, 70 kb, 80 kb, 90 kb, 100 kb, 110 kb, 120 kb, 130 kb, 140 kb, 150 kb, 160 kb, 170 kb, 180 kb, 190 kb, 200 kb, 210 kb, 220 kb, 230 kb, 240 kb, 250 kb, 260 kb, 270 kb, 280 kb, 290 kb, 300 kb, 350 kb, 400 kb, 450 kb, 500 kb, or 1000 kb of nucleotides. In some embodiments, there is no disruption in the animal's endogenous lambda light chain immunoglobulin gene.

In some embodiments, the deleted sequence starts from IGLV2 to IGLC1, from IGLV3 to IGLC1, or from IGLJ2 to IGLC1.

Genetically Modified Animals

In one aspect, the present disclosure provides genetically-modified, non-human animal comprising a humanized heavy chain immunoglobulin locus and/or a humanized light chain immunoglobulin locus. In some embodiments, the animal comprises one or more human IGHV genes, one or more human IGHD genes, one or more human IGHJ genes, one or more human IGKV genes and/or one or more human IGKJ genes. In some embodiments, these genes are at the endogenous immunoglobulin gene locus.

In some embodiments, the animal comprises a human lambda chain immunoglobulin locus. In some embodiments, the animal comprises a disruption in the animal's endogenous lambda light chain immunoglobulin gene locus. In some embodiments, the animal does not have a disruption in the animal's endogenous lambda light chain immunoglobulin gene locus.

The genetically modified non-human animal can be various animals, e.g., a mouse, rat, rabbit, pig, bovine (e.g., cow, bull, buffalo), deer, sheep, goat, chicken, cat, dog, ferret, primate (e.g., marmoset, rhesus monkey). For the non-human animals where suitable genetically modifiable embryonic stem (ES) cells are not readily available, other methods are employed to make a non-human animal comprising the genetic modification. Such methods include, e.g., modifying a non-ES cell genome (e.g., a fibroblast or an induced pluripotent cell) and employing nuclear transfer to transfer the modified genome to a suitable cell, e.g., an oocyte, and gestating the modified cell (e.g., the modified oocyte) in a non-human animal under suitable conditions to form an embryo. These methods are known in the art, and are described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2003, which is incorporated by reference herein in its entirety. Thus, in various embodiments, human V, D, and/or J segments can be operably linked to non-human animal (e.g., rodent, mouse, rat, hamster) constant region gene sequences. During B cell development, these rearranged human V, D, and/or J segments are linked to the non-human animal immunoglobulin constant region.

In one aspect, the animal is a mammal, e.g., of the superfamily Dipodoidea or Muroidea. In some embodiments, the genetically modified animal is a rodent. The rodent can be selected from a mouse, a rat, and a hamster. In some embodiments, the genetically modified animal is from a family selected from Calomyscidae (e.g., mouse-like hamsters), Cricetidae (e.g., hamster, New World rats and mice, voles), Muridae (true mice and rats, gerbils, spiny mice, crested rats), Nesomyidae (climbing mice, rock mice, with-tailed rats, Malagasy rats and mice), Platacanthomyidae (e.g., spiny dormice), and Spalacidae (e.g., mole rates, bamboo rats, and zokors). In some embodiments, the genetically modified rodent is selected from a true mouse or rat (family Muridae), a gerbil, a spiny mouse, and a crested rat. In some embodiments, the non-human animal is a mouse.

In some embodiments, the animal is a mouse of a C57 background (e.g., a C57BL strain selected from C57BL/A, C57BL/An, C57BL/GrFa, C57BL/KaLwN, C57BL/6, C57BL6J, C57BL/6ByJ, C57BL/6NJ, C57BL/10, C57BL/10ScSn, C57BL/10Cr, and C57BL/O1a). In some embodiments, the mouse is a 129 strain selected from the group consisting of a strain that is 129P1, 129P2, 129P3, 129X1, 129S1 (e.g., 129S1/SV, 129S1/SvIm), 129S2, 129S4, 12955, 129S9/SvEvH, 129S6 (129/SvEvTac), 129S7, 129S8, 129T1, 129T2. These mice are described, e.g., in Festing et al., Revised nomenclature for strain 129 mice, Mammalian Genome 10: 836 (1999); Auerbach et al., Establishment and Chimera Analysis of 129/SvEv- and C57BL/6-Derived Mouse Embryonic Stem Cell Lines (2000), both of which are incorporated herein by reference in the entirety. In some embodiments, the genetically modified mouse is a mix of the 129 strain and the C57BL/6 strain. In some embodiments, the mouse is a mix of the 129 strains, or a mix of the BL/6 strains. In some embodiments, the mouse is a BALB strain, e.g., BALB/c strain. In some embodiments, the mouse is a mix of a BALB strain and another strain. In some embodiments, the mouse is from a hybrid line (e.g., 50% BALB/c-50% 12954/Sv; or 50% C57BL/6-50% 129).

In some embodiments, the animal is a rat. The rat can be selected from a Wistar rat, an LEA strain, a Sprague Dawley strain, a Fischer strain, F344, F6, and Dark Agouti. In some embodiments, the rat strain is a mix of two or more strains selected from the group consisting of Wistar, LEA, Sprague Dawley, Fischer, F344, F6, and Dark Agouti.

The animal can have one or more other genetic modifications, and/or other modifications, that are suitable for the particular purpose for which the humanized animal is made.

Genetically modified non-human animals that comprise a modification of an endogenous non-human immunoglobulin gene locus. In some embodiments, the modification can comprise a human nucleic acid sequence encoding at least a portion of a human protein (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human heavy chain variable domain or light chain variable domain sequence). Although genetically modified cells are also provided that can comprise the modifications described herein (e.g., ES cells, somatic cells), in many embodiments, the genetically modified non-human animals comprise the modification of the endogenous locus in the germline of the animal.

Genetically modified animals can express a humanized antibody and/or a chimeric antibody from endogenous mouse loci, wherein one or more endogenous mouse immunoglobulin genes have been replaced with human immunoglobulin genes and/or a nucleotide sequence that is at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical to the human immunoglobulin gene sequences (e.g., IGHV, IGHD, IGHJ, IGKV and/or IGKJ genes). In various embodiments, an endogenous non-human immunoglobulin gene locus is modified in whole or in part to comprise human nucleic acid sequence.

Genetic, molecular and behavioral analyses for the non-human mammals described above can performed. The present disclosure also relates to the progeny produced by the non-human mammal provided by the present disclosure mated with the same or other genotypes. Non-human mammals can be any non-human animal known in the art and which can be used in the methods as described herein. Preferred non-human mammals are mammals, (e.g., rodents). In some embodiments, the non-human mammal is a mouse.

The present disclosure also provides a cell line or primary cell culture derived from the non-human mammal or a progeny thereof. A model based on cell culture can be prepared, for example, by the following methods. Cell cultures can be obtained by way of isolation from a non-human mammal, alternatively cell can be obtained from the cell culture established using the same constructs and the standard cell transfection techniques. The integration of genetic constructs containing DNA sequences encoding human or humanized immunoglobulins can be detected by a variety of methods.

There are many analytical methods that can be used to detect exogenous DNA or modifications on the genomic DNA, including methods at the level of nucleic acid (including the mRNA quantification approaches using reverse transcriptase polymerase chain reaction (RT-PCR) or Southern blotting, and in situ hybridization) and methods at the protein level (including histochemistry, immunoblot analysis and in vitro binding studies). In addition, the expression level of the gene of interest can be quantified by ELISA techniques well known to those skilled in the art. Many standard analysis methods can be used to complete quantitative measurements. For example, transcription levels can be measured using RT-PCR and hybridization methods including RNase protection, Southern blot analysis, RNA dot analysis (RNAdot) analysis. Immunohistochemical staining, flow cytometry, Western blot analysis can also be used to assess the presence of human or humanized proteins.

Antibodies and Antigen Binding Fragments

The present disclosure provides antibodies and antigen-binding fragments thereof (e.g., humanized antibodies or chimeric antibodies) that are produced by the methods described herein.

In general, antibodies (also called immunoglobulins) are made up of two classes of polypeptide chains, light chains and heavy chains. A non-limiting antibody of the present disclosure can be an intact, four immunoglobulin chain antibody comprising two heavy chains and two light chains. The heavy chain of the antibody can be of any isotype including IgM, IgG, IgE, IgA, or IgD or subclasses including IgG1, IgG2, IgG2a, IgG2b, IgG3, IgG4, IgE1, IgE2, etc. The light chain can be a kappa light chain or a lambda light chain. An antibody can comprise two identical copies of a light chain and two identical copies of a heavy chain. The heavy chains, which each contain one variable domain (or variable region, V_(H)) and multiple constant domains (or constant regions), bind to one another via disulfide bonding within their constant domains to form the “stem” of the antibody. The light chains, which each contain one variable domain (or variable region, VL) and one constant domain (or constant region), each bind to one heavy chain via disulfide binding. The variable region of each light chain is aligned with the variable region of the heavy chain to which it is bound. The variable regions of both the light chains and heavy chains contain three hypervariable regions sandwiched between more conserved framework regions (FR).

These hypervariable regions, known as the complementary determining regions (CDRs), form loops that comprise the principle antigen binding surface of the antibody. The four framework regions largely adopt a beta-sheet conformation and the CDRs form loops connecting, and in some cases forming part of, the beta-sheet structure. The CDRs in each chain are held in close proximity by the framework regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding region.

Methods for identifying the CDR regions of an antibody by analyzing the amino acid sequence of the antibody are well known, and a number of definitions of the CDRs are commonly used. The Kabat definition is based on sequence variability, and the Chothia definition is based on the location of the structural loop regions. These methods and definitions are described in, e.g., Martin, “Protein sequence and structure analysis of antibody variable domains,” Antibody engineering, Springer Berlin Heidelberg, 2001. 422-439; Abhinandan, et al. “Analysis and improvements to Kabat and structurally correct numbering of antibody variable domains,” Molecular immunology 45.14 (2008): 3832-3839; Wu, T. T. and Kabat, E. A. (1970) J. Exp. Med. 132: 211-250; Martin et al., Methods Enzymol. 203:121-53 (1991); Morea et al., Biophys Chem. 68(1-3):9-16 (October 1997); Morea et al., J Mol Biol. 275(2):269-94 (January 1998); Chothia et al., Nature 342(6252):877-83 (December 1989); Ponomarenko and Bourne, BMC Structural Biology 7:64 (2007); each of which is incorporated herein by reference in its entirety.

The CDRs are important for recognizing an epitope of an antigen. As used herein, an “epitope” is the smallest portion of a target molecule capable of being specifically bound by the antigen binding domain of an antibody. The minimal size of an epitope may be about three, four, five, six, or seven amino acids, but these amino acids need not be in a consecutive linear sequence of the antigen's primary structure, as the epitope may depend on an antigen's three-dimensional configuration based on the antigen's secondary and tertiary structure.

In some embodiments, the antibody is an intact immunoglobulin molecule (e.g., IgG1, IgG2a, IgG2b, IgG3, IgG4, IgM, IgD, IgE, IgA). The IgG subclasses (IgG1, IgG2, IgG3, and IgG4) are highly conserved, differ in their constant region, particularly in their hinges and upper CH2 domains. The sequences and differences of the IgG subclasses are known in the art, and are described, e.g., in Vidarsson, et al, “IgG subclasses and allotypes: from structure to effector functions.” Frontiers in immunology 5 (2014); Irani, et al. “Molecular properties of human IgG subclasses and their implications for designing therapeutic monoclonal antibodies against infectious diseases.” Molecular immunology 67.2 (2015): 171-182; Shakib, Farouk, ed. The human IgG subclasses: molecular analysis of structure, function and regulation. Elsevier, 2016; each of which is incorporated herein by reference in its entirety.

The antibody can also be an immunoglobulin molecule that is derived from any species (e.g., human, rodent, mouse, rat, camelid). Antibodies disclosed herein also include, but are not limited to, polyclonal, monoclonal, monospecific, polyspecific antibodies, and chimeric antibodies that include an immunoglobulin binding domain fused to another polypeptide. The term “antigen binding domain” or “antigen binding fragment” is a portion of an antibody that retains specific binding activity of the intact antibody, i.e., any portion of an antibody that is capable of specific binding to an epitope on the intact antibody's target molecule. It includes, e.g., Fab, Fab′, F(ab′)2, and variants of these fragments. Thus, in some embodiments, an antibody or an antigen binding fragment thereof can be, e.g., a scFv, a Fv, a Fd, a dAb, a bispecific antibody, a bispecific scFv, a diabody, a linear antibody, a single-chain antibody molecule, a multi-specific antibody formed from antibody fragments, and any polypeptide that includes a binding domain which is, or is homologous to, an antibody binding domain. Non-limiting examples of antigen binding domains include, e.g., the heavy chain and/or light chain CDRs of an intact antibody, the heavy and/or light chain variable regions of an intact antibody, full length heavy or light chains of an intact antibody, or an individual CDR from either the heavy chain or the light chain of an intact antibody.

In some embodiments, the antigen binding fragment can form a part of a chimeric antigen receptor (CAR). In some embodiments, the chimeric antigen receptor are fusions of single-chain variable fragments (scFv) as described herein, fused to CD3-zeta transmembrane- and endodomain.

In some embodiments, the scFV has one heavy chain variable domain, and one light chain variable domain. In some embodiments, the scFV has two heavy chain variable domains, and two light chain variable domains. In some embodiments, the scFV has two antigen binding regions, and the two antigen binding regions can bind to the respective target antigens.

The antibodies and antigen-binding fragments thereof (e.g., humanized antibodies or chimeric antibodies) that are produced by the methods described herein have various advantages. In some embodiments, no further optimization is required to obtain desired properties (e.g., binding affinities, thermal stabilities, and/or limited aggregation).

In some implementations, the antibody (or antigen-binding fragments thereof) specifically binds to a target with a dissociation rate (koff) of less than 0.1 s⁻¹, less than 0.01 s⁻¹, less than 0.001 s⁻¹, less than 0.0001 s⁻¹, or less than 0.00001 s⁻¹. In some embodiments, the dissociation rate (koff) is greater than 0.01 s⁻¹, greater than 0.001 s⁻¹, greater than 0.0001 s⁻¹, greater than 0.00001 s⁻¹, or greater than 0.000001 s⁻¹.

In some embodiments, kinetic association rates (kon) is greater than 1×10²/Ms, greater than 1×10³/Ms, greater than 1×10⁴/Ms, greater than 1×10⁵/Ms, or greater than 1×10⁶/Ms. In some embodiments, kinetic association rates (kon) is less than 1×10⁵/Ms, less than 1×10⁶/Ms, or less than 1×10⁷/Ms.

Affinities can be deduced from the quotient of the kinetic rate constants (KD=koff/kon). In some embodiments, KD is less than 1×10⁻⁶ M, less than 1×10⁻⁷ M, less than 1×10⁻⁸ M, less than 1×10⁻⁹ M, or less than 1×10⁻¹⁰ M. In some embodiments, the KD is less than 50 nM, 40 nM, 30 nM, 20 nM, 15 nM, 10 nM, 9 nM, 8 nM, 7 nM, 6 nM, 5 nM, 4 nM, 3 nM, 2 nM, or 1 nM. In some embodiments, KD is greater than 1×10⁻⁷M, greater than 1×10⁻⁸ M, greater than 1×10⁻⁹ M, greater than 1×10⁻¹⁰ M, greater than 1×10⁻¹¹ M, or greater than 1×10⁻¹² M. In some embodiments, the antibody binds to a target with KD less than or equal to about 0.9 nM, 0.8 nM, 0.7 nM, 0.6 nM, 0.5 nM, 0.4 nM, 0.3 nM, 0.2 nM, or 0.1 nM.

In some embodiments, thermal stabilities are determined. The antibodies or antigen binding fragments as described herein can have a Tm greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.

As IgG can be described as a multi-domain protein, the melting curve sometimes shows two transitions, or three transitions, with a first denaturation temperature, Tm D1, and a second denaturation temperature Tm D2, and optionally a third denaturation temperature Tm D3.

In some embodiments, the antibodies or antigen binding fragments as described herein has a Tm D1 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C. In some embodiments, the antibodies or antigen binding fragments as described herein has a Tm D2 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C. In some embodiments, the antibodies or antigen binding fragments as described herein has a Tm D3 greater than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.

In some embodiments, Tm, Tm D1, Tm D2, Tm D3 are less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.

In some embodiments, the antibodies or antigen binding fragments as described herein do not form aggregation when the temperate is less than 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, or 95° C.

Methods of Making Genetically Modified Animals

The genetically modified animals can be made by introducing human immunoglobulin genes into the genome of non-human animals to produce animals that can express humanized antibodies or chimeric antibodies. FIG. 1A shows the methods of making the humanized animals. In some embodiments, the methods first involve modifying the human immunoglobulin locus on the human chromosome. The modified human chromosomes are then introduced into the mouse recipient cell. The human immunoglobulin variable region is then introduced into the corresponding region of the mouse genome by direct replacement. Then, the recipient cells are screened. In some embodiments, the cells do not contain the human chromosomes. The cells are then injected to blastocysts to prepare chimeric mice. Subsequent breeding can be performed to obtain mice containing intact humanized immunoglobulin locus.

Several other techniques may be used in making genetically modified animals, including, e.g., nonhomologous end-joining (NHEJ), homologous recombination (HR), zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALEN), and the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system. In some embodiments, homologous recombination is used. In some embodiments, CRISPR-Cas9 genome editing is used to generate genetically modified animals. Many of these genome editing techniques are known in the art, and is described, e.g., in Yin et al., “Delivery technologies for genome editing,” Nature Reviews Drug Discovery 16.6 (2017): 387-399, which is incorporated by reference in its entirety. Many other methods are also provided and can be used in genome editing, e.g., micro-injecting a genetically modified nucleus into an enucleated oocyte, and fusing an enucleated oocyte with another genetically modified cell.

The genetic modification process can involve replacing endogenous sequence with human sequence by homologous recombination. In some embodiments, the cleavage at the upstream and the downstream of the target site (e.g., by zinc finger nucleases, TALEN or CRISPR) can result in DNA double strands break, and the homologous recombination is used to replace endogenous sequence with human sequence.

In some embodiments, the methods for making a genetically modified, humanized animal, can include the step of replacing at an endogenous locus (or site), a nucleic acid (e.g., V, D, J regions, or V, J regions) with a corresponding region of human sequence. The sequence can include a region (e.g., a part or the entire region) of IGHV, IGHD, IGHJ, IGKV, and/or IGKJ genes. In some embodiments, the replacement is mediated by homologous recombination. In some embodiments, the replacement is mediated by Cre recombinase.

FIG. 9 shows a targeting strategy for adding functional genetic elements into the human chromosome. These vectors can be inserted at the upstream of the V region, between the J region and the C region.

In some embodiments, the first vector has from 5′ to 3′ one or more of the following: DNA homology arm sequence at upstream of the insertion site, PGK promoter, red fluorescent protein reporter gene (tdTomato), FMDV (Foot-And-Mouth Disease Viruses) self-cleaving peptide (2A), zeomycin resistance gene (Zeo), transcription termination/polyadenylation signal sequence (PolyA; “PA”), the LoxP recognition sequence, hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; “3′HygR”), and the Flp recognition target (“FRT”), downstream DNA homology arm sequence, and DTA gene.

The second vector has from 5′ to 3′ one or more of the following: DNA homology arm sequence at the upstream of the insertion site, the LoxP recognition sequence, PGK promoter, a partial sequence of Puromycin resistance gene (5′PuroR), a mammalian expression promoter (EF-1a) from human elongation factor 1 alpha, piggyBac transposase gene sequence (PBase), an internal Ribosomal Entry Sites (IRES), kanamycin resistance gene sequence (Neo), transcription termination/polyadenylation signal sequence, DNA homology arm sequence at the downstream of insertion site, and DTA.

These vectors can be integrated into the genome of the cells, and the cells can be selected by drug resistance markers or a combination thereof (e.g., Zeocin, G418, and/or Puromycin). In some embodiments, the PB transposase is expressed, and the genetic elements between the transposase target sequence can be deleted.

In some embodiments, these vectors are integrated into a human chromosome that has been modified. The human chromosome can be modified first, before the first and the second vectors are integrated into the genome. In some embodiments, one or more additional vectors can be added at various locations of the chromosome as needed. In some embodiments, the vector is added between the C region and the centromere. The third vector can have from 5′ to 3′ one or more of the following: DNA homology arm sequence at the upstream of the insertion site, PGK promoter, Puromycin resistance gene sequence (PuroR), thymidine kinase gene sequence (TK), the LoxP recognition sequence, PGK promoter, puromycin resistance gene partial sequence (5′PuroR), a mammalian expression promoter (EF-1a), PBase, IRES, Neo, transcription termination/polyadenylation signal sequence, DNA homology arm sequence at the downstream of insertion site, and DTA. In some embodiments, these vectors can be inserted into the variable gene region or constant region. In some embodiments, a part of endogenous variable gene region or endogenous constant region is deleted. In some embodiments, a large fragment of the chromosome is deleted (e.g., between the constant region and the centromere). The cells can also be treated with Cre enzyme, leading to the recombination of the loxP sites, thereby removing genomic DNA sequences between the J region and the centromere on human chromosome 14 or between the C region and the centromere on human chromosome 14. In some embodiments, spontaneous chromosome breakage can occur. Modified human chromosomes with desired chromosome breakage can be selected for experiments.

The human chromosome can be obtained from human cell lines, cancer cells, primary cell culture, and/or human fibroblasts. In some embodiments, the human cell is introduced with a first vector and is then fused with a recipient cell. The modified chromosome is then separated and introduced into another appropriate recipient cell. Cells with the desired resistance are selected to obtain cells containing only one human chromosome. Then, a second vector is introduced into the cells, and the cells are selected by resistance. Then, if needed, a third vector, and/or a fourth vector can be introduced. The recipient cell can be a mammalian cell, a human cell, or a mouse cell. In some embodiments, the recipient cell is a CHO cell, or preferably an A9 cell. In some embodiments, the modified chromosomes are labeled by fluorescence and separated. And the modified chromosomes are injected into the recipient cells by chromosome microinjection. In some embodiments, the donor cells are induced to multinucleate their chromosomes. These nuclei are then forced through the cell membrane to create microcells, which can be fused to a recipient cell. In some embodiments, microcell-mediated chromosome transfer can also be used. The chromosome manipulation techniques are described e.g., in CN1200014A; CN109837307A; US20120093785A1; and US2009253902; Kuroiwa et al. “Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts.” Nature Biotechnology 18.10 (2000)): 1086-1090; Chinese patent CN1717483A; Paulis, Marianna. “Chromosome Transfer Via Cell Fusion.” Methods in Molecular Biology 738(2011):57; Genes, Chromosomes & Cancer 14: 126127 (1995); Tomizuka et al. “Functional expression and germline atransmission of a human chromosome fragment in chimaeric mice.” Nature Genetics 16.2 (1997): 133-143; Somatic Cell and Molecular Genetics, Vol. 13, No. 3, 1987, pp. 279-284; each of which is incorporated herein by reference in its entirety.

In some embodiments, the modification can be made to the mouse chromosome. The targeting strategy is shown in FIG. 4 . The first vector can have DNA homology arm sequence at upstream and downstream of the insertion site, and a LoxP sequence. In some embodiments, the first vector has from the 5′ to 3′ one or more of the following: DNA homology arm sequence at upstream of the insertion site, Flp recognition target (FRT), CAG promoter, hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; “5′HygR”), LoxP, FRT, 5′ PB transposon sequence (PB5′), PGK promoter, blue fluorescent protein reporter gene (BFP), FMDV self-cleaving peptide (2A), hygromycin resistance gene (hygromycin phosphotransferase; HygR), 3′ PB transposon sequence (PB3′), the DNA homology arm sequence at downstream of the insertion site, and DTA.

The second vector can have DNA homology arm sequence at upstream and downstream of the insertion site, and LoxP sequence. In some embodiments, the second vector has from 5′ to 3′ one or more of the following: DNA homology arm sequence at upstream of the insertion site, 5′ PB transposon sequence (PB5′), PGK promoter, green fluorescent protein reporter gene sequence (EGFP), FMDV self-cleaving peptide (2A), Puromycin resistance gene sequence (PuroR), 3′ PB transposon sequence (PB3′), Flp recognition target (FRT), puromycin resistance gene partial sequence (3′PuroR), FMDV self-cleaving peptide (2A), DT receptor (DTR), LoxP recognition sequence, DNA homology arm sequence at downstream of insertion site, and DTA.

FIG. 30 show a similar targeting strategy for kappa light chain immunoglobulin locus. Two vectors can first be integrated to human chromosome. The first vector has DNA homology arm sequences at upstream and downstream of the insertion site, and LoxP recognition sequence. In some embodiments, the first vector has from 5′ to 3′ one or more of the following: DNA homology arm sequence at upstream of the insertion site, PGK promoter, tdTomato, FMDV self-cleaving peptide (2A), Bsr, termination of transcription/polyadenylation signal sequence, LoxP recognition sequence, hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; “3′HygR”), FRT, the DNA homology arm sequence at downstream of the insertion site, and DTA.

The second vector has DNA homology arm sequences at upstream and downstream of the insertion site, and LoxP recognition sequence. In some embodiments, the second vector has from 5′ to 3′ one or more of the following: the DNA homology arm sequence at upstream of the insertion site, the LoxP recognition sequence, the PGK promoter, a portion of puromycin resistance gene sequence (5′PuroR), EF-1a, PBase, IRES, Neo, transcription termination/polyadenylation signal sequence, DNA homology arm sequence at downstream of insertion site, and DTA.

LoxP recognition sequences can also be added to the human chromosome (e.g., human chromosome 2, 14, 22). The cells can also be treated with Cre enzyme, leading to the recombination of the loxP sites, thereby removing genomic DNA sequences. In some embodiments, spontaneous chromosome breakage can be used to remove genomic DNA sequences as well.

The modification on mouse light chain immunoglobulin locus can be directly performed. In some embodiments, a vector is directly used to replace the entire mouse light chain immunoglobulin variable region. In some embodiments, the vector has from the 5′ to 3′: DNA homology arm sequence at upstream of the insertion site, Flp recognition target (FRT), mammalian expression promoter (EF-1a) from human elongation factor 1 alpha, hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; “5′HygR”), the LoxP recognition sequence for the Cre recombinase, 5′ PB transposon sequence (PB5′), blue fluorescent protein reporter gene (BFP), DT receptor (DTR), FMDV self-cleaving peptide (2A), kanamycin resistance gene sequence (Neo), transcription termination/polyadenylation signal sequence (PolyA; “PA”), 3′ PB transposon sequence (PB3′), puromycin resistance gene partial sequence (3′PuroR), FMDV self-cleaving peptide (2A), DT receptor (DTR), the LoxP recognition sequence for the Cre recombinase, DNA homology arm sequence at downstream of insertion site, and DTA.

The mouse immunoglobulin variable region can be replaced by the human immunoglobulin variable region by replacement (e.g., homologous recombination, or Cre mediated recombination). In some embodiments, Cre recombination can be used to mediate the replacement. In some embodiments, the vectors can add LoxP recognition sequence into the human chromosome. Similar modifications can be made to the mouse chromosome, wherein two LoxP recognition sequences can be added to the chromosome. For example, Cre recombinase can then mediate the replacement of V, J regions on mouse chromosome with the V, J regions on human chromosome or the replacement of V, D, J regions on mouse chromosome with the V, D, J regions on human chromosome.

The cells can be further screened for cells that do not have human chromosomes (e.g., by DT). In some cases, cells that are not screened by DT may contain recombinant human chromosome fragments, but these fragments are small and are unstable in mouse cells (e.g., Shinohara et al. (2000) Chromosome Research, 8: 713-725), and will naturally disappear during cell proliferation. In some embodiments, a large fragment of the modified human chromosome is deleted, e.g., by Cre-mediated deletion or by spontaneous chromosomal breakage.

The 5′ end homology arm and/or the 3′ end homology arm can have a desired length to facilitate homologous recombination. In some embodiments, the homology arm is about or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 kb (e.g., about 3 kb). In some embodiments, the homology arm is less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, or 50 kb.

In some embodiments, the vector may also optionally include a reporter protein, e.g., a luciferase (e.g., Gluc) or a fluorescent protein (e.g., EGFP, BFP, etc.).

These modifications can be performed in various cells. In some embodiments, the cell is a stem cell, an embryonic stem cell, or a fertilized egg cell.

The present disclosure further provides a method for establishing a humanized animal model, involving the following steps:

(a) providing the cell (e.g. a fertilized egg cell) based on the methods described herein;

(b) culturing the cell in a liquid culture medium;

(c) transplanting the cultured cell to the fallopian tube or uterus of the recipient female non-human mammal, allowing the cell to develop in the uterus of the female non-human mammal;

(d) identifying the germline transmission in the offspring genetically modified humanized non-human mammal of the pregnant female in step (c).

In some embodiments, the non-human mammal in the foregoing method is a mouse (e.g., a C57 mouse, a BALB/c mouse, or a C57BL/6 mouse).

In some embodiments, the non-human mammal in step (c) is a female with pseudo pregnancy (or false pregnancy).

In some embodiments, the fertilized eggs for the methods described above are C57BL6 fertilized eggs. Other fertilized eggs that can also be used in the methods as described herein include, but are not limited to, FVB/N fertilized eggs, BALB/c fertilized eggs, DBA/1 fertilized eggs and DBA/2 fertilized eggs.

Fertilized eggs can come from any non-human animal, e.g., any non-human animal as described herein. In some embodiments, the fertilized egg cells are derived from rodents. The genetic construct can be introduced into a fertilized egg by microinjection of DNA. For example, by way of culturing a fertilized egg after microinjection, a cultured fertilized egg can be transferred to a false pregnant non-human animal, which then gives birth of a non-human mammal, so as to generate the non-human mammal mentioned in the methods described above.

Cells, tissues, and animals (e.g., mouse) are also provided that comprise the nucleotide sequences as described herein, as well as cells, tissues, and animals (e.g., mouse) that express humanized or chimeric antibodies from an endogenous non-human locus.

The present disclosure also provides various targeting vectors (e.g., vectors that are useful for making the genetically modified animals). In some embodiments, the vector can comprise: a) a DNA fragment homologous to the 5′ end of a region to be altered (5′ homology arm); b) a sequence comprising desired genetic elements (e.g., LoxP recognition site, drug resistance genes, and/or reporter genes etc.); and c) a second DNA fragment homologous to the 3′ end of the region to be altered (3′ homology arm). The disclosure also relates to a cell comprising the targeting vectors as described herein.

In some embodiments, the genes in the cell are heterozygous. In some embodiments, the genes in the cell are homozygous.

In some embodiments, the non-human mammalian cell is a mouse cell. In some embodiments, the cell is a fertilized egg cell.

The present disclosure further relates to methods for generating genetically modified animal model with two or more human or chimeric genes. The animal can comprise one or more human or humanized immunoglobulin locus and a sequence encoding an additional human or chimeric protein. In some embodiments, the additional human or chimeric protein can be programmed cell death protein 1 (PD-1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), Lymphocyte Activating 3 (LAG-3), B And T Lymphocyte Associated (BTLA), Programmed Cell Death 1 Ligand 1 (PD-L1), CD27, CD28, CD47, CD137, CD154, T-Cell Immunoreceptor With Ig And ITIM Domains (TIGIT), T-cell Immunoglobulin and Mucin-Domain Containing-3 (TIM-3), Glucocorticoid-Induced TNFR-Related Protein (GITR), or TNF Receptor Superfamily Member 4 (TNFRSF4 or OX40).

The methods of generating genetically modified animal model with additional human or chimeric genes (e.g., humanized genes) can include the following steps:

(a) using the methods as described herein to obtain a genetically modified non-human animal;

(b) mating the genetically modified non-human animal with another genetically modified non-human animal, and then screening the progeny to obtain a genetically modified non-human animal with two or more human or chimeric genes.

In some embodiments, in step (b) of the method, the genetically modified animal can be mated with a genetically modified non-human animal with human or chimeric PD-1, CTLA-4, LAG-3, BTLA, PD-L1, CD27, CD28, CD47, CD137, CD154, TIGIT, TIM-3, GITR, SIRPa, or OX40. Some of these genetically modified non-human animal are described, e.g., in PCT/CN2017/090320, PCT/CN2017/099577, PCT/CN2017/099575, PCT/CN2017/099576, PCT/CN2017/099574, PCT/CN2017/106024, PCT/CN2017/110494, PCT/CN2017/110435, PCT/CN2017/120388, PCT/CN2018/081628, PCT/CN2018/081629; each of which is incorporated herein by reference in its entirety.

In some embodiments, the genetically modified animals can have a human ADAM6 gene, an endogenous ADAM6 gene or a modified ADAM6 gene. The ADAM6 protein is a member of the ADAM family of proteins, where ADAM is an acronym for A Disintegrin And Metalloprotease. The human ADAM6 gene, normally found between human IGHV genes IGHV1-2 and IGHV6-1, is a pseudogene (FIG. 37 ). In mice, there are two ADAM6 genes, ADAM6a and ADAM6b. They are located in an intergenic region between mouse IGHV and IGHD gene clusters. The mouse ADAM6a is located between mouse IGHV5-1 and mouse IGHD5-1. The mouse ADAM6b is located between mouse IGHD3-1 and mouse IGHD1-1. Thus, in some embodiments, the genetically modified animals can have a human ADAM6 gene. In some embodiments, the genetically modified animals do not have an endogenous ADAM6 gene.

In some embodiments, the genetically modified animals are mice. In some embodiments, the mice are modified to include a nucleotide sequence that encodes an ADAM6 protein (e.g., ADAM6a or ADAM6b). In some embodiments, the sequence is placed at any suitable position. It can be placed in the intergenic region, or in any suitable position in the genome. In some embodiments, the nucleic acid encodes a sequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to a mouse ADAM6a gene (e.g., 113539230-113547024 of NC_000078.6; SEQ ID NO: 53) or a mouse ADAM6b gene (e.g., 113486188-113492125 of NC_000078.6; SEQ ID NO: 54). In some embodiments, the nucleic acid additionally includes the regulatory elements for the ADAM6a gene and ADAM6b gene (e.g., promoters).

In some embodiments, a functional mouse ADAM6 locus can be placed in the midst of human IGHV gene cluster. In some embodiment, the mouse ADAM6 locus is between two human IGVH genes. In some embodiments, the human ADAM6 pseudogene between human VH1-2 and human VH(II)-1-1 is replaced with the mouse ADAM6 locus. In some embodiments, the ADAM6a gene and the ADAM6b gene are located between human IGHV1-2 and human VH(II)-1-1 in the genome of the animal. In some embodiments, the location of the mouse ADAM6 sequence within the human gene sequence can approximate the position of the human ADAM6 pseudogene or can approximate the position of the mouse ADAM6 sequence (e.g., within the V-D intergenic region). In some embodiments, the genetic modified mice has a humanized heavy chain immunoglobulin locus. In some embodiments, the mouse ADAM6a and the mouse ADAM6b are located between human IGHV1-2 and IGHV6-1 genes. Placing the mouse ADAM6a and the mouse ADAM6b between human IGHV1-2 and IGHV6-1 genes can have various advantages. For example, because these genes replaces the human ADAM6 gene at the same locus, it is likely that the replacement of human ADAM6 gene will have limited impact on the VDJ recombination and the mouse ADAM6a and the mouse ADAM6b gene can also function properly (as in a location that is similar to the endogenous locus).

Thus, in one aspect, the disclosure provides a genetically-modified animal comprising at an endogenous heavy chain immunoglobulin gene locus, a first sequence comprising one or more human IGHV genes; a second sequence comprising a ADAM6 gene; and a third sequence comprising one or more human IGHD genes, and one or more human IGHJ genes. In some embodiments, the first sequence, the second sequence, and the third sequence are operably linked.

In some embodiments, the first sequence comprises all human IGHV genes in Table 1 except IGHV2-10, IGHV3-9, IGHV1-8, IGHV(II)-1-1, and IGHV6-1. In some embodiments, the first sequence comprises all human IGHV genes in Table 1 except IGHV5-10-1 and IGHV3-64D, IGHV(II)-1-1, and IGHV6-1. In some embodiments, the first sequence is an unmodified sequence derived from a human heavy chain immunoglobulin gene locus.

In some embodiments, the second sequence comprises either one or both of a mouse ADAM6a gene and a mouse ADAM6b gene. In some embodiments, the animal is a fertile male mouse. In some embodiments, the second sequence does not have a mouse ADAM6a gene or a mouse ADAM6b gene.

In some embodiments, the third sequence comprises all human IGHD genes in Table 2, and all human IGHJ genes in Table 3. In some embodiments, the third sequence comprises human IGHV6-1. In some embodiments, the third sequence comprises human IGHV(II)-1-1. In some embodiments, the third sequence is an unmodified sequence derived from a human heavy chain immunoglobulin gene locus.

In some embodiments, the AMAM6a and/or ADAM6b are endogenous sequences. In some embodiments, the AMAM6a and/or ADAM6b are not replaced, and/or located in its endogenous or native position. In some embodiments, the mouse IGHV genes before mouse IGHV1-2 in the heavy chain variable region locus are replaced with human IGHV genes. In some embodiments, the mouse IGHV, IGHD and IGHJ genes after mouse IGHV6-1 in the heavy chain variable region locus are replaced with one or more human IGHV genes, IGHD and/or IGHJ genes.

Thus, in some embodiments, the mouse IGHV, IGHD and IGHJ genes can be replaced with human IGHV, IGHD and IGHJ by more than one replacements. In the first step, a selected number of mouse IGHV genes on the 5′ side of the ADAM6a (e.g., all mouse IGHV genes in Table 4) are replaced with human IGHV genes. In the second step, a selected number of mouse IGHD and IGHJ genes on the 3′ side of the ADAM6b (e.g., all mouse IGHD genes in Table 5 except IGHD5-1 and IGHD3-1 and all IGHJ genes in Table 6) are replaced with human IGHD and human IGHJ genes. The replacement can be performed by homologous recombination or Cre-mediated recombination.

In some embodiments, the mice do not have mouse ADAM6a or ADAM6b genes. In some embodiments, the mice have human ADAM6 genes.

Various methods can be used to increase the fertility of the mice. In some embodiments, female mice with superovulation can be used in mating. In some embodiments, in vitro fertilization can be used. Superovulation can be induced by injecting serum gonadotropin and chorionic gonadotropin (e.g., human or mouse CG) into a mature female mouse. A mature male mouse can be sacrificed and its cauda epididymides can be isolated. The duct of cauda epididymis is cut open to release sperm. Next, a superovulating mature female mouse can be sacrificed and the oviducts can be isolated. Cumulus-oocyte-complexes (COCs) can be released from the oviduct. Next, sperm suspension can be added to the COCs and incubated for insemination. Pathenogenic oocytes containing only one pronucleus can be removed. After the incubation, embryos at 2-cell stage can be transferred to recipient females. Methods of increasing mouse fertility are known in the art.

The disclosure also provides a nucleic acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any nucleotide sequence as described herein, and an amino acid sequence that is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identical to any amino acid sequence as described herein.

In some embodiments, the disclosure relates to nucleotide sequences encoding any peptides that are described herein, or any amino acid sequences that are encoded by any nucleotide sequences as described herein. In some embodiments, the nucleic acid sequence is less than 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150, 200, 250, 300, 350, 400, 500, or 600 nucleotides. In some embodiments, the amino acid sequence is less than 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, or 400 amino acid residues.

In some embodiments, the amino acid sequence (i) comprises an amino acid sequence; or (ii) consists of an amino acid sequence, wherein the amino acid sequence is any one of the sequences as described herein.

In some embodiments, the nucleic acid sequence (i) comprises a nucleic acid sequence; or (ii) consists of a nucleic acid sequence, wherein the nucleic acid sequence is any one of the sequences as described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid “identity” is equivalent to amino acid or nucleic acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present invention, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The percentage of residues conserved with similar physicochemical properties (percent homology), e.g. leucine and isoleucine, can also be used to measure sequence similarity. Families of amino acid residues having similar physicochemical properties have been defined in the art. These families include e.g., amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The homology percentage, in many cases, is higher than the identity percentage. Thus, the present disclosure also provides an amino acid sequence that has at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% homology percentage to any amino acid sequence as described herein, or a nucleic acid encoding these amino acid sequences.

Methods of Using Genetic Modified Animals

The genetic modified animals can be used to generate humanized or chimeric antibodies that can bind specifically to a target. In some embodiments, the target (e.g., a protein or a fragment of the protein) can be used as an immunogen to generate antibodies in these animals using standard techniques for polyclonal and monoclonal antibody preparation. In some embodiments, the genetic modified animal is exposed to a selected antigen for a time and under conditions which permit the animal to produce antibody specific for the antigen.

Polyclonal antibodies can be raised in animals by multiple injections (e.g., subcutaneous or intraperitoneal injections) of an antigenic peptide or protein. In some embodiments, the antigenic peptide or protein is injected with at least one adjuvant. In some embodiments, the antigenic peptide or protein can be conjugated to an agent that is immunogenic in the species to be immunized. Animals can be injected with the antigenic peptide or protein more than one time (e.g., twice, three times, or four times).

The full-length polypeptide or protein can be used or, alternatively, antigenic peptide fragments thereof can be used as immunogens. The antigenic peptide of a protein comprises at least 8 (e.g., at least 10, 15, 20, or 30) amino acid residues of the amino acid sequence and encompasses an epitope of the protein such that an antibody raised against the peptide forms a specific immune complex with the protein.

An immunogen typically is used to prepare antibodies by immunizing a suitable subject (e.g., the genetically modified animal as described herein). An appropriate immunogenic preparation can contain, for example, a recombinantly-expressed or a chemically-synthesized polypeptide (e.g., a fragment of the protein). The preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or a similar immunostimulatory agent.

Polyclonal antibodies can be prepared as described above by immunizing a suitable subject with a polypeptide, or an antigenic peptide thereof (e.g., part of the protein) as an immunogen. The antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme-linked immunosorbent assay (ELISA) using the immobilized polypeptide or peptide. If desired, the antibody molecules can be isolated from the mammal (e.g., from the blood) and further purified by well-known techniques, such as protein A of protein G chromatography to obtain the IgG fraction. At an appropriate time after immunization, e.g., when the specific antibody titers are highest, antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler et al. (Nature 256:495-497, 1975), the human B cell hybridoma technique (Kozbor et al., Immunol. Today 4:72, 1983), the EBV-hybridoma technique (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96, 1985), or trioma techniques. The technology for producing hybridomas is well known (see, generally, Current Protocols in Immunology, 1994, Coligan et al. (Eds.), John Wiley & Sons, Inc., New York, N.Y.). Hybridoma cells producing a monoclonal antibody are detected by screening the hybridoma culture supernatants for antibodies that bind the polypeptide or epitope of interest, e.g., using a standard ELISA assay.

In one aspect, the disclosure provides a mouse that comprises a modification of an endogenous immunoglobulin heavy chain locus, wherein the mouse produces a B cell that comprises a rearranged immunoglobulin sequence operably linked to a heavy chain constant region gene sequence. In some embodiment, the rearranged immunoglobulin sequence operably linked to the heavy chain constant region gene sequence comprises a human heavy chain V, D, and/or J sequence. In some embodiments, the heavy chain constant region gene sequence comprises a human or a mouse heavy chain sequence selected from the group consisting of a CH1, a hinge, a CH2, a CH3, and a combination thereof.

In one aspect, the disclosure provides a mouse that comprises a modification of an endogenous immunoglobulin light chain (e.g., kappa or lambda) locus, wherein the mouse produces a B cell that comprises a rearranged immunoglobulin sequence operably linked to a light chain constant region gene sequence. In some embodiments, the rearranged immunoglobulin sequence operably linked to the light chain constant region gene sequence comprises a human light chain V and/or J sequence. In some embodiments, the light chain constant region gene sequence comprises a human or a mouse light chain constant region.

The mouse B cells or spleen cells can comprise a rearranged non-mouse immunoglobulin variable gene sequence, e.g., operably linked to a mouse immunoglobulin constant region gene. The sequences for encoding human heavy chain variable region and human light chain variable region are determined. The sequences can be determined by e.g., sequencing the hybridoma of interest or B cells. In some embodiments, single B cell screening is used. It can screen the natural antibody repertoire without the need for hybridoma fusion and combinatorial display. For example, B cells can be mixed with a panel of DNA-barcoded antigens, such that both the antigen barcode(s) and B-cell receptor (BCR) sequences of individual B cells are recovered via single-cell sequencing protocols.

The antibodies can be further modified to obtain a humanized antibody or a human antibody, e.g., by operably linking the sequence encoding human heavy chain variable region to a sequence encoding a human heavy chain constant region, and/or operably linking the sequence encoding human light chain variable region to a sequence encoding a human light chain constant region.

In some embodiments, if the mouse expresses a protein that is very similar to the antigen of interest, it can be difficult to elicit an immune response in the mouse. This is because during immune cell development, B-cells and T-cells that recognize MHC molecules bound to peptides of self-origin are deleted from the repertoire of immune cells. In those cases, the humanized mouse can be further modified. The corresponding gene in the mouse can be knocked out, and the mouse is then exposed to the antigen of interest. Because the mouse does not go through negative selection for the gene product, the mouse can generate an antibody that can specifically bind to the target easily.

The disclosure also provides methods of making antibodies, nucleic acids, cells, tissues (e.g., spleen tissue). In some embodiments, the methods involve exposing the animal as described herein to the antigen. Antibodies (e.g., hybrid antibodies), nucleic acids encoding the antibodies, cells, and/or tissues (e.g., spleen tissue) can be obtained from the animal. In some embodiments, the nucleic acids encoding human heavy and light chain immunoglobulin variable regions are determined, e.g., by sequencing. In some embodiments, the nucleic acid encoding the human heavy chain immunoglobulin variable region can be operably linked with a nucleic acid encoding a human heavy chain immunoglobulin constant region. In some embodiments, the nucleic acid encoding the human light chain immunoglobulin variable region can be operably linked with a nucleic acid encoding a human light chain immunoglobulin constant region. In some embodiments, the cells containing the nucleic acids as described herein are cultured and the antibodies are collected.

In some embodiments, no mouse immunoglobulin V, D, J genes (e.g., no mouse IGHV, IGHD, IGHJ, IGKV, or IGKJ genes) contributes to the heavy chain and/or light chain variable region sequence. In some embodiments, the heavy chain and/or light chain variable region sequence produced by the animal are fully human, and are completely contributed by human immunoglobulin V, D, J genes (e.g., human IGHV, IGHD, IGHJ, IGKV, and IGKJ genes).

Variants of the antibodies or antigen-binding fragments described herein can be prepared by introducing appropriate nucleotide changes into the DNA encoding a human, humanized, or chimeric antibody, or antigen-binding fragment thereof described herein, or by peptide synthesis. Such variants include, for example, deletions, insertions, or substitutions of residues within the amino acids sequences that make-up the antigen-binding site of the antibody or an antigen-binding domain. In a population of such variants, some antibodies or antigen-binding fragments will have increased affinity for the target protein. Any combination of deletions, insertions, and/or combinations can be made to arrive at an antibody or antigen-binding fragment thereof that has increased binding affinity for the target. The amino acid changes introduced into the antibody or antigen-binding fragment can also alter or introduce new post-translational modifications into the antibody or antigen-binding fragment, such as changing (e.g., increasing or decreasing) the number of glycosylation sites, changing the type of glycosylation site (e.g., changing the amino acid sequence such that a different sugar is attached by enzymes present in a cell), or introducing new glycosylation sites.

Antibodies disclosed herein can be derived from any species of animal, including mammals. Non-limiting examples of native antibodies include antibodies derived from humans, primates, e.g., monkeys and apes, cows, pigs, horses, sheep, camelids (e.g., camels and llamas), chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies.

Human and humanized antibodies include antibodies having variable and constant regions derived from (or having the same amino acid sequence as those derived from) human germline immunoglobulin sequences. Human antibodies may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs.

Additional modifications to the antibodies or antigen-binding fragments can be made. For example, a cysteine residue(s) can be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have any increased half-life in vitro and/or in vivo. Homodimeric antibodies with increased half-life in vitro and/or in vivo can also be prepared using heterobifunctional cross-linkers as described, for example, in Wolff et al. (Cancer Res. 53:2560-2565, 1993). Alternatively, an antibody can be engineered which has dual Fc regions (see, for example, Stevenson et al., Anti-Cancer Drug Design 3:219-230, 1989).

In some embodiments, a covalent modification can be made to the antibody or antigen-binding fragment thereof. These covalent modifications can be made by chemical or enzymatic synthesis, or by enzymatic or chemical cleavage. Other types of covalent modifications of the antibody or antibody fragment are introduced into the molecule by reacting targeted amino acid residues of the antibody or fragment with an organic derivatization agent that is capable of reacting with selected side chains or the N- or C-terminal residues.

EXAMPLES

The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

Example 1: Overview

Experiments were performed to introduce human immunoglobulin genes into the mouse genome to produce mice expressing humanized antibodies. FIG. 1A shows the methods of making the humanized mice. The methods first involve modifying the human immunoglobulin region on the human chromosome. The modified human chromosomes were then introduced into the mouse recipient cell.

The mouse immunoglobulin variable region was replaced by the human immunoglobulin variable region by direct replacement (e.g., homologous recombination, or Cre mediated recombination). In some cases, the human immunoglobulin variable region can be introduced into the mouse genome by a stepwise approach. Then, the recipient cells were screened for the correct replacement. The cells were then injected to blastocysts to prepare chimeric mice. Subsequent breeding was performed to obtain mice containing intact human immunoglobulin variable regions.

Because the mouse heavy chain gene and the two light chain genes are located on chromosomes 12, 6, and 16, respectively, mice containing the human heavy chain variable region or the human light chain variable region can be prepared separately (FIGS. 1B and 1C). These mice can then be mated with each other to obtain mice that can express both the human heavy chain variable domain and the human light chain variable domain.

Example 2: Modification of the Mouse Heavy Chain Immunoglobulin Locus

The heavy chain immunoglobulin locus is located on mouse chromosome 12. FIG. 2 is a schematic diagram showing the mouse heavy chain immunoglobulin locus. Two recombination sites (1301, 1302) were introduced on both sides of the variable region of the heavy chain immunoglobulin locus, and the resulting modified chromosome is shown in FIG. 3A-3B. One of them is a wildtype loxP site, the other is a heterospecific mutant lox site (lox2272). The recombination cannot occur between the wildtype loxP site and the heterospecific mutant lox site. The modification was performed in mouse embryonic stem cells. An overview of the targeting strategy is shown in FIG. 4 . The vector (V1401) has from the 5′ to 3′: DNA homology arm sequence at upstream of the insertion site, Flp recognition target (FRT), CAG promoter, hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; “5′HygR”), LoxP (1301), FRT, 5′ PB transposon sequence (PB5′), PGK promoter, blue fluorescent protein reporter gene (BFP), FMDV self-cleaving peptide (2A), hygromycin resistance gene (hygromycin phosphotransferase; HygR), 3′ PB transposon sequence (PB3′), the DNA homology arm sequence at downstream of the insertion site, and DTA.

The vector (V1402) has from 5′ to 3′: DNA homology arm sequence at upstream of the insertion site, 5′ PB transposon sequence (PB5′), PGK promoter, green fluorescent protein reporter gene sequence (EGFP), FMDV self-cleaving peptide (2A), Puromycin resistance gene sequence (PuroR), 3′ PB transposon sequence (PB3′), Flp recognition target (FRT), puromycin resistance gene partial sequence (3′PuroR), FMDV self-cleaving peptide (2A), DT receptor (DTR), LoxP recognition sequence (1302), DNA homology arm sequence at downstream of insertion site, and DTA.

The vectors (V1401 and V1402) were introduced into mouse embryonic stem cells. The cells were then screened by hygromycin B and puromycin. The integration of the exogenous genes into mouse genomes was confirmed by PCR. The results are shown in FIGS. 5A-5B and FIGS. 6A-6B. The clones numbered 030, 035, 036 and 037 were confirmed to be positive.

The PCR assay was performed using the following primers:

mIgHV-5′loxP-L-GT-F: (SEQ ID NO: 1) 5′-gccaaggaatttaaaaggggattgaaagcaa-3′, mIGHV-005-L-GT-R2: (SEQ ID NO: 2) 5′-gccctccatgtacagcttcatgtgc-3′; mIGHV-005-5′loxP-R-GT-F2: (SEQ ID NO: 3) 5′-actgggcttgtcgagacagagaaag-3′, mIgHV-5′loxP-R-GT-R: (SEQ ID NO: 4) 5′-ccacagcccgatctacttggctttt-3′; mIGHV-3′lox-L-GT-F2: (SEQ ID NO: 5) 5′-gcaaggttttgactaagcggagcac-3′; mIGHV-3′lox-L-GT-R2: (SEQ ID NO: 6) 5′-tgacgcatgtgttttatcggtctgt-3′; mIGHV3′lox-R-GT-F2: (SEQ ID NO: 7) 5′-gtgcctgacacgtgctacgagattt-3′; mIGHV-3′lox-R-GT-R1: (SEQ ID NO: 8) 5′-ttcaacaataagcagggccagaggg-3′;

Among these primers, mIgHV-5′loxP-L-GT-F and mIgHV-5′loxP-R-GT-R are located on the mouse chromosome, mIGHV-005-L-GT-R2 and mIGHV-005-5′loxP-R-GT-F2 are located on the vector 1401, mIGHV-3′lox-L-GT-F2 and mIGHV-3′lox-R-GT-R1 are located on the mouse chromosome, mIGHV-3′lox-L-GT-R2 and mIGHV3′lox-R-GT-F2 are located on the vector 1402.

Example 3: Modifying Human Chromosome 14

The purpose of the experiment is to generate a modified human chromosome with at least two recombination sites. The two recombination sites were introduced on both sides of the variable region of the heavy chain immunoglobulin locus.

The heavy chain immunoglobulin locus is located on human chromosome 14. FIG. 7 is a schematic diagram of the human chromosome 14 highlighting the heavy chain immunoglobulin locus.

The modified human variable region is shown in FIG. 8 . An overview of the targeting strategy is shown in FIG. 9 . As shown in FIG. 9 , the 301 and 302 sites are recombination sites. The recombination site 1301 and the recombination site 301 are identical. The recombination site 1302 and the recombination site 302 are identical.

Experiments were performed to insert vectors into human chromosome 14 at the upstream of the V region, and between the J region and the C region. The first targeting vector (V401) from 5′ to 3′ has DNA homology arm sequence at upstream of the insertion site, PGK promoter, red fluorescent protein reporter gene (tdTomato), self-cleaving peptide (2A) from FMDV (Foot-And-Mouth Disease Viruses), Zeocin resistance gene sequence (Zeo), transcription termination/polyadenylation signal sequence (PolyA; “PA”), the LoxP recognition sequence (301) for the Cre recombinase, hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; “3′HygR”), and the Flp recognition target (“FRT”), downstream DNA homology arm sequence, and diphtheria toxin subunit A (DTA) gene.

The second vector (V402) from 5′ to 3′ has the following: DNA homology arm sequence at the upstream of the insertion site, the LoxP recognition sequence (302) for the Cre recombinase, PGK promoter, a partial sequence of Puromycin resistance gene (5′PuroR), a mammalian expression promoter (EF-1a) from human elongation factor 1 alpha, piggyBac transposase gene sequence (PBase), an internal Ribosomal Entry Sites (IRES), kanamycin resistance gene sequence (Neo), transcription termination/polyadenylation signal sequence (PolyA; “PA”), DNA homology arm sequence at the downstream of insertion site, and DTA.

In some experiments, the vectors (V401, V402) were introduced into the cells, and the cells were selected by appropriate drug resistance markers or a combination thereof (Zeocin, G418).

There are many ways to introduce the vectors of interest into the human chromosome. The human chromosome can be obtained from human cell lines, cancer cells, primary cell culture, and/or human fibroblasts. In one experiment, the first vector is introduced into the chromosome. The modified chromosome can be added to the recipient cell, and then the second vector can be inserted to the modified chromosome. In some experiments, V401 was first introduced into human cells, and then the chromosome was labelled by fluoresce and was then separated, and the modified chromosome was then injected into recipient cells by microinjection. V402 was then introduced into the cells. In another experiment, the human fibroblasts were selected and were introduced with vector 402. The human fibroblasts were then fused with recipient cells (A9 cells or CHO cells).

In some experiments, one or more vectors can be inserted into human chromosome 14 at the desired locations by homologous recombination e.g., in the same time. The vectors can contain drug resistance markers (Zeocin, G418) and the cells are then screened. The chromosome is the labelled and is then separated, and the modified chromosome is then injected into recipient cells by chromosome microinjections.

In some experiments, one or more additional vectors are inserted. These additional vectors can be inserted at different locations of human chromosome 14 as needed. In one experiment, a third vector can have from 5′ to 3′ the following: DNA homology arm sequence at the upstream of the insertion site, PGK promoter, Puromycin resistance gene sequence (PuroR), thymidine kinase gene sequence (TK), the LoxP recognition sequence (302), PGK promoter, puromycin resistance gene partial sequence (5′PuroR), a mammalian expression promoter (EF-1a) from human elongation factor 1 alpha, piggyBac transposase gene sequence (PBase), Internal Ribosomal Entry Sites (IRES), kanamycin resistance gene sequence (Neo), transcription termination/polyadenylation signal sequence (PolyA; “PA”), DNA homology arm sequence at the downstream of insertion site, and DTA. The vector is inserted within the C region.

In one experiment, a third vector (V403) was inserted between the C region and the kinetochore. The vector from 5′ to 3′ has the following: DNA homology arm sequence at the upstream of the insertion site, PGK promoter, Puromycin resistance gene sequence (PuroR), thymidine kinase gene sequence (TK), the LoxP recognition sequence (302), PGK promoter, puromycin resistance gene partial sequence (5′PuroR), a mammalian expression promoter (EF-1a) from human elongation factor 1 alpha, piggyBac transposase gene sequence (PBase), Internal Ribosomal Entry Sites (IRES), kanamycin resistance gene sequence (Neo), transcription termination/polyadenylation signal sequence (PolyA; “PA”), DNA homology arm sequence at the downstream of insertion site, and DTA.

In one experiment, the human fibroblasts were selected and were introduced with vector 402. The human fibroblasts were then fused with recipient cells (A9 cells or CHO cells). The modified chromosome was separated and introduced into another appropriate recipient cell. Cells were then selected by G418 resistance to obtain cells containing only one human chromosome. Then, vector 401 was introduced into the cells, and the cells were selected by resistance to Zeocin. After that, vector 403 was introduced in the cells, and the cells were selected by resistance to puromycin. The positive clones selected after screening were treated with Cre enzyme. The chromosome techniques were described e.g., in Kuroiwa et al. “Manipulation of human minichromosomes to carry greater than megabase-sized chromosome inserts.” Nature Biotechnology 18.10 (2000)): 1086-1090; CN1200014A; CN109837307A; US20120093785A1; US2009253902; CN1717483A; Paulis, Marianna. “Chromosome Transfer Via Cell Fusion.” Methods in Molecular Biology 738(2011):57; Genes, Chromosomes & Cancer 14: 126127 (1995); Tomizuka et al. “Functional expression and germline a transmission of a human chromosome fragment in chimaeric mice.” Nature Genetics 16.2 (1997): 133-143; and Somatic Cell and Molecular Genetics, Vol. 13, No. 3, 1987, pp. 279-284; each of which is incorporated herein by reference in its entirety.

PCR was performed to confirm the presence of the 5′-end recombination site 301 and the 3′-end recombination site 302 on the chromosome. Cells without random insertion were confirmed by Southern Blot and were analyzed by fluorescence in situ hybridization (FISH).

FIG. 10 shows the modified human chromosome 14. FIG. 11 shows the results of PCR identification of loxP site 301 on chromosome hChr14-mut3. FIG. 12 shows the results of PCR identification of loxP site 302 on chromosome hChr14-mut3. As shown in the figures, 12 clones (numbered 1-B2, 1-B8, 1-D6, 1-D10, 1-F11, 1-G11, 2-A2, 3-E5, 3-G5, 3-H4, 5-C3, and 6-F11) were positive clones.

The following PCR primers were used in the experiments:

hIGHV-5′loxP-L-GT-F1: (SEQ ID NO: 9) 5′-TCAAAGTCAATTTCCTCAGCGAGGCT-3′, hIGHV-5′loxP-R-GT-R: (SEQ ID NO: 10) 5′-AGGGAGGGAATGGAATGAGGGTGAT-3′; hIGHV-3′loxP-L-GT-F1: (SEQ ID NO: 11) 5′-CCATGTGACCCATTCGAGTGTCCTG-3′, hIGHV-3′loxP-R-GT-R: (SEQ ID NO: 12) 5′-TTGTGAGGGCTCAAGTTCAGTGCAT-3′.

FISH analysis was performed using the positive clones using CCP14 FISH Probe (CytoTest Inc., Rockville, Md., catalog number CT-CCP014). The representative FISH images for the clone 1-D10 are shown in FIGS. 13 and 14 . In FIG. 13 , the white arrow indicates the full length of the human chromosome 14 (before the modification). In FIG. 14 , the white arrow indicates the modified human chromosome 14 fragment.

Example 4: Introduction of Human Chromosomes or Fragment into Mouse ES Cells

The modified chromosome obtained in Example 3 was introduced into cell obtained Example 2 by methods described previously. The cells were then screened by G418. Only the cell containing only one human chromosome was selected. FIG. 15 shows the modified mouse chromosome 12.

Cre recombinase then mediated the replacement of V, D, J regions on mouse chromosome mChr12-mut2 with the V, D, J regions on human chromosome hChr14-mut3 (FIG. 16 ). The human chromosome DNA sequence was replaced by the sequence between the recombination sites 1301 and 1302. Hygromycin and puromycin were used for screening positive cells. The cells were further screened by DT to obtain mouse cells that do not contain human chromosomes before being injected into mouse blastocysts. In some cases, the cells were directly injected into blastocysts without DT screening.

The cells after Cre recombination were tested to confirm that the human gene sequences were integrated into the mouse genome. The PCR results are shown in FIGS. 17, 18, 19, and 20 . All PCR results showed that cells numbered 1-B4, 1-B10, and 2-A7 had the correct recombination, and the human chromosomes in 1-B10 cells disappeared. Murine Whole Chromosome Painting Probes (Cytocell Ltd, Cambridge, UK; Cat. No. AMP12R) and human-specific IGH Breakapart Probes (Cytocell Ltd, Cambridge, UK; Cat. No. LPH 014) were used to test 1-B10 cells by FISH. The result is shown in FIG. 21 , confirming that human chromosome fragments were present in the mouse chromosome. These primers are shown in the table below.

TABLE 12 Product size NO. Primer Sequence (5′-3′) (bp) 1 M5-L F gccaaggaatttaaaaggggattg 4227 aaagcaa (SEQ ID NO: 13) R cgagagctgtggagagaaaggcaa a (SEQ ID NO: 14) 2 M5-R F tatgtcctgcgggtaaatagctgc 3109 g (SEQ ID NO: 15) R agggagggaatggaatgagggtga t (SEQ ID NO: 16) 3 M3 F ccatgtgacccattcgagtgtcct 3738 g (SEQ ID NO: 17) R cttaccatttgcggtgcctggttt c (SEQ ID NO: 18) 4 H5-L F tcaaagtcaatttcctcagcgagg 5329 ct (SEQ ID NO: 19) R aggaattgtatcccataggctagc acgt (SEQ ID NO: 20) 5 H5-R F gacgacgtgaccctgttcatcagc 2863 (SEQ ID NO: 21) R ccacagcccgatctacttggcttt t (SEQ ID NO: 22) 6 H3-L F gcaaggttttgactaagcggagca 4098 c (SEQ ID NO: 23) R cactagtctcgtgcagatggacag c (SEQ ID NO: 24) 7 H3-R F gttagaagacttcctctgccctcg 8120 g (SEQ ID NO: 25) R ttgtgagggctcaagttcagtgca t (SEQ ID NO: 26)

Example 5: Making Mice Containing a Humanized Heavy Chain Immunoglobulin Locus

The positive clone cells were injected into the blastocysts of BALB/c mice by microinjection. The embryo microinjection was carried out according to the method described, e.g., in A. Nagy, et al., “Manipulating the Mouse Embryo: A Laboratory Manual (Third Edition),” Cold Spring Harbor Laboratory Press, 2003. The injected fertilized eggs were then transferred to a culture medium for a short time culture, and then was transplanted into the oviduct of the recipient mouse to produce the genetically modified humanized mice (F0 generation). The mice were then mated with mice having C57BL/6 background. The black progeny were selected to mate with Flp tool mice (FIG. 22 ). PCR analysis was performed on the DNA obtained from the tail of the mice. The mice were further crossed with mice with BALB/c background several times (e.g., at least 5 times) to obtain humanized heavy chain immunoglobulin locus heterozygous mice with BALB/c background.

In order to confirm that the mouse expresses the human antibody heavy chain, blood was collected from the mice of the chimeric mouse (F0 generation) and the black mouse (F1 generation). The RNA was extracted and reverse-transcribed to obtain cDNA. The following PCR primer were used to amplify the sequence, and the sequences were further sequenced.

TABLE 13 PCR primers NO. Primer Sequence (5′-3′) 1 VH1 F CAGGTSCAGCTGGTRCAGTC (SEQ ID NO: 27) R AGGGATCCAGAGTTCCAGGT (SEQ ID NO: 28) 2 VH2 F CAGRTCACCTTGAAGGAGTC (SEQ ID NO: 29) R AGGGATCCAGAGTTCCAGGT (SEQ ID NO: 28) 3 VH3 F SAGGTGCAGCTGGTGGAGTC (SEQ ID NO: 30) R AGGGATCCAGAGTTCCAGGT (SEQ ID NO: 28) 4 VH4 F CAGGTGCAGCTGCAGGAGTC (SEQ ID NO: 31) R AGGGATCCAGAGTTCCAGGT (SEQ ID NO: 28) 5 VH5 F GARGTGCAGCTGGTGCAGTC (SEQ ID NO: 32) R AGGGATCCAGAGTTCCAGGT (SEQ ID NO: 28) 6 VH6 F CAGGTACAGCTGCAGCAGTC (SEQ ID NO: 33) R AGGGATCCAGAGTTCCAGGT (SEQ ID NO: 28)

Example 6: Modification of the Mouse Light Chain Immunoglobulin Locus

The light chain immunoglobulin locus is located on mouse chromosome 6. FIG. 23 was a schematic diagram showing the mouse light chain immunoglobulin locus. Two recombination sites were introduced on both sides of the variable region of the light chain immunoglobulin locus, and the resulting modified chromosome was shown in FIGS. 24A-24B. The detailed targeting strategy were shown in FIG. 25 .

The modification was performed in mouse embryonic stem cells. The vector (V3901) had from the 5′ to 3′: DNA homology arm sequence at upstream of the insertion site, Flp recognition target (FRT), mammalian expression promoter (EF-1a) from human elongation factor 1 alpha, hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; “5′HygR”), the LoxP recognition sequence (1101) for the Cre recombinase, 5′ PB transposon sequence (PB5′), blue fluorescent protein reporter gene (BFP), DT receptor (DTR), FMDV self-cleaving peptide (2A), kanamycin resistance gene sequence (Neo), transcription termination/polyadenylation signal sequence (PolyA; “PA”), 3′ PB transposon sequence (PB3′), puromycin resistance gene partial sequence (3′PuroR), FMDV self-cleaving peptide (2A), DT receptor (DTR), the LoxP recognition sequence (1102) for the Cre recombinase, DNA homology arm sequence at downstream of insertion site, and DTA.

The vectors (V3901) was introduced into mouse embryonic stem cells. The cells were screened by corresponding antibiotic resistance gene markers, or their combinations. The integration of the vector V3901 into correct locus in mouse genomes was confirmed by PCR. The results were shown in FIGS. 26-27 . The two combined PCR results confirmed that the cells numbered 208, 209, 215, 217 and 269 were positive clones.

The PCR assay was performed using the following primers:

IGKV-005-C-5G-L-GT-F:  (SEQ ID NO: 34) 5′-TCACACACTACAGCTTCCACCACAA-3′; IGKV-005-C-5G-L-GT-R2:  (SEQ ID NO: 35) 5′-CGGGGAAAAGTCGACTCTAGAACGG-3′; IGKV-005-C-5G-R-GT-F1:  (SEQ ID NO: 36) 5′-ACTGCATTCTAGTTGTGGTTTGTCCA-3′; IGKV-005-C-5G-R-GT-R:  (SEQ ID NO: 37) 5′-GGCCTGGAAAACTCAGCTATCCTTT-3′.

Among these primers, IGKV-005-C-5G-L-GT-F and IGKV-005-C-5G-R-GT-R were located on the mouse chromosome, IGKV-005-C-5G-L-GT-R2 and IGKV-005-C-5G-R-GT-F1 were located on the vector V3901.

Thus, two recombination sites were introduced into mouse chromosome 6 in mouse embryonic stem cells.

Example 7: Modifying Human Chromosome 2

The human light chain immunoglobulin locus is located in human chromosome 2. FIG. 28 is a schematic diagram of the human chromosome 2 highlighting the light chain immunoglobulin locus.

Two recombination sites were introduced on both sides of the variable region of the light chain immunoglobulin locus. The region between V_(HK) and the centromere (kinetochore) was deleted to obtain a shorter artificial chromosome for subsequent experiments. Similar recombination sites were introduced into the variable region of the mouse immunoglobulin locus on chromosome 6. Then the human chromosome was introduced into the mouse recipient cell to obtain a humanized light chain immunoglobulin locus.

The modified human chromosome 2 is shown in FIG. 29 . The targeting strategy is shown in FIG. 30 . The vector (V2701) has from 5′ to 3′: DNA homology arm sequence at upstream of the insertion site, PGK promoter, red fluorescent protein reporter gene sequence (tdTomato), FMDV self-cleaving peptide (2A), Blasticidin S deaminase (Bsr) from Aspergillus terreus, termination of transcription/polyadenylation signal sequence (PolyA; “PA”), LoxP recognition sequence 2601, hygromycin resistance gene (partial sequence of hygromycin phosphotransferase; “3′HygR”), Flp recognition target (FRT), the DNA homology arm sequence at downstream of the insertion site, and diphtheria toxin subunit A (DTA).

The vector (V2702) has from 5′ to 3′: the DNA homology arm sequence at upstream of the insertion site, the LoxP recognition sequence 2602, the PGK promoter, a portion of puromycin resistance gene sequence (5′PuroR), EF-1a, PBase, IRES, kanamycin resistance gene sequence (Neo), transcription termination/polyadenylation signal sequence (PolyA; “PA”), DNA homology arm sequence at downstream of insertion site, and DTA.

The sequence of vector (V2702) was verified by sequencing. The vector was introduced into human H9 cells by transfection. The cells were then screened by G418 and Ouabain resistance. The integration of the genes into human genomes was confirmed by PCR. The results are shown in FIG. 31 . The clones numbered 01, 02, 03, and 04 were confirmed to be positive clones.

The PCR assay was performed using the following primers:

3′L-L-GT-F: (SEQ ID NO: 38) 5′-AAGGTGACTCTGCAATCAGCCTCTG-3′, 3′L-L-GT-R1: (SEQ ID NO: 39) 5′-TCATCTACAGCCACAACGTGAGCAG-3′; 3′L-R-GT-F1: (SEQ ID NO: 40) 5′-CCCATGTACAGGTTCCGCATGAACT-3′, 3′L-R-GT-R: (SEQ ID NO: 41) 5′-CTCCGTCCGCTTTTATTTCCCCTGT-3′.

Cells with modified chromosomes that were suitable for further experiments were selected. The modified human chromosome was introduced into recipient cells by chromosome technique. The recipient cells A9 cells were screened for G418 resistance. The cells containing only one human chromosome were selected for further gene editing.

During the screening, the clone numbered 03 shown in FIG. 31 had the correct recombination and was labeled by a human chromosome 2 counting probe (CCP2 FISH Probe) (CytoTest Inc., Rockville, Md., catalog number CT-CCP002). The result confirmed that there was a modified human chromosome 2 in the cell (FIG. 32 ).

The vector (V2701) was then further introduced into the cells. The cells were screened by G418 and Blasticidin S resistance. The recombination was confirmed by PCR. The primers are shown in the table below. The results showed that 5-C3, 1-H2, 1-H9 and 1-G5 were positive clones (FIG. 33 ).

TABLE 14 Product size NO. Primer Sequence (5′-3′) (bp) 1 hIgK3 F GTTATAACACGGGGAGTGCGTGT 4501 GC (SEQ ID NO: 42) R GTTTGGACAAACCACAACTAGAA TGCAGTG (SEQ ID NO: 43) 2 hIgK4 F GCAACGGCTACAATCAACAGCAT 3196 CC (SEQ ID NO: 44) R TGGGTCTGGGACAGACTTCACTC TC (SEQ ID NO: 45) 3 hIgK1 F AAGGTGACTCTGCAATCAGCCTC 4623 TG (SEQ ID NO: 46) R TCATCTACAGCCACAACGTGAGC AG (SEQ ID NO: 47) 4 hIgK2 F CCCATGTACAGGTTCCGCATGAA 4470 CT (SEQ ID NO: 48) R CTCCGTCCGCTTTTATTTCCCCT GT (SEQ ID NO: 49)

Example 8: Making Mice that can Produce Humanized Antibodies

The mouse embryonic stem cells and the cells obtained in Example 7 were fused and the modified human chromosome 2 was introduced into mouse embryonic stem cell obtained in Example 6. The mouse ES cells containing only one modified human chromosome 2 was selected, and the cells were screened after Cre-mediated recombination (Cre mediated recombination) was shown in FIG. 34 . The cells after Cre recombination were tested to confirm that the human gene sequences were integrated into the mouse genome. Mouse Whole Chromosome Painting Probes (Cytocell Ltd, Cambridge, UK; Cat. No. AMP06G) and human-specific IGK Breakapart Probes (Cytocell Ltd, Cambridge, UK; Cat. No. LPH 034) were used to verify PCR-confirmed positive clones by fluorescence in situ hybridization (FISH). The result was shown in FIG. 35 , confirming that human chromosome fragments were present in the mouse chromosome. The cells were injected into the blastocysts. Mice containing the humanized light chain immunoglobulin locus can be obtained.

The chimeric mice were selected to mate with C57BL/6 mice, producing gray and black progeny (F1 generation). PCR analysis was performed on the DNA obtained from the tail of the black mice, and positive F1 generation mice were selected to mate with Flp tool mice. FIG. 36 showed a schematic diagram of the Flp-mediated recombination. The mice prepared by the methods contained C57BL/6 background. Mice with different background can have different advantages, and the heterozygous or homozygous mice prepared by the methods herein can be used to generate mice with some other background by backcrossing (for example, BALB/c mice have a humoral immune advantage) for several generations to obtain mice with the desired backgrounds.

A few mice were selected and crossed with BALB/c mice several times to obtain heterozygous mice with BALB/c background. The heterozygous mice then were then crossed with each other to obtain homozygous mice.

The mice with humanized light chain immunoglobulin locus and the mice with humanized heavy chain immunoglobulin locus were crossed with each other to obtain mice with both humanized heavy chain immunoglobulin locus and humanized light chain immunoglobulin locus.

Example 9: B-Cell Development in Transgenic Mice

Experiments were performed to compare the immune systems of the humanized mice and the wild-type mice. Three 9-10 week old wild-type (WT), three mice with heterozygous humanized heavy chain immunoglobulin locus, and three mice with homozygous humanized heavy chain immunoglobulin locus were selected. Among them, the heterozygous mice and the homozygous mice had similar body weight, appearance and vitality as compared to the wild-type mice. Peripheral blood, spleen, lymph nodes and bone marrow tissues of these mice were obtained, and no obvious anatomical changes were discovered (for example, there was no observable difference of spleen size, morphology and weight among the three groups of mice). Flow cytometry was performed to analyze lymphocyte populations and distribution in the peripheral blood, spleen, and lymph nodes (FIGS. 45-47 ) and B cell populations in the spleen, lymph nodes, and bone marrow (FIGS. 48-50 ) of the mice. In the results, the leukocytes included: B cells (e.g., characterized by CD45+, CD19+, TCR−), T cells, and natural killer (NK) cells (e.g., characterized by CD45+, TCR−, NK1.1+). T cells were characterized by CD45+, CD19−, TCR+. CD4+ T Cells (CD4) were characterized by CD45+, CD19−, TCR+, CD4+, CD8−. And CD8+ T cells (CD8) were characterized by CD45+, CD19−, TCR+, CD4−, CD8+. Only intact, single, live leukocytes were included in the flow cytometry analysis.

The development stages of B cells in lymph nodes and spleen are categorized into T1 (Transitional type 1 B cell, characterized by B220⁺IgM⁺IgD⁻), T2 (Transitional type 2 B cell, characterized by B220⁺IgM⁺IgD⁺) and mature B cells (characterized by B220⁺IgM^(low)IgD⁺). FIGS. 45-47 show percentages of leukocytes in different tissue samples. FIGS. 48-49 indicated percentages of B cells at different developmental stages.

In addition, B cell development were also evaluated at the spleen marginal zone (Marginal-zone B cell, MZ-B, characterized by B220^(low)CD21^(high)CD23^(low)) and follicular zone (Follicular B cell, referred to as FO-B, characterized by B220+CD21^(low)CD23⁺). FIG. 50 shows percentages of splenic B cells at spleen marginal zone (MZ-B) and follicular zone (FO-B).

Based on different developmental stages, B cells in the bone marrow were categorized into pro-B-cells (characterized by B220^(low)CD43^(high)IgM^(low)), pre-B-cells (characterized by B220^(low)CD43^(int)IgM^(low)) and immature-B-cells (characterized by B220^(high)CD43^(low)IgM^(high)). FIGS. 51A-51C indicated percentages of B cells at different developmental stages in bone marrows.

Compared with the wild-type mice, percentages of immune cells and B cells in humanized mice were similar and there was no statistical difference among different groups. No significant defects in B cell differentiation were observed in either heavy chain humanized heterozygous mice (heterozygote) or homozygous mice (homozygote).

Example 10: Serum Immunoglobulin Isotype Analysis

Further, the levels of various immunoglobulins in the serum of the unimmunized mice in the Example above were analyzed. The mice included WT mice, mice with heterozygous humanized heavy chain immunoglobulin locus, and mice with homozygous humanized heavy chain immunoglobulin locus.

The experiments were performed using the Clonotyping System-B6/C57J-HRP (Southern Biotech, Cat #5300-05B) kit. First, the capture antibody Goat Anti-Mouse Ig, Human ads-UNLB was diluted to 10 ug/mL with PBS (Solarbio, Cat #P1020). Then, 0.1 mL of the diluted antibody was added to each well of the enzyme-linked immunosorbent assay (ELISA) plate and incubated at 37° C. for 2 hours. Next, the plate was washed and blocked at 4° C. for 12 hours. The serum samples were serially diluted with 1% BSA (Cell Signaling, Cat #9998). 0.1 mL of the diluted samples were added to each well, followed by incubation at 37° C. for 1 hour. 1% BSA was added to a well as a blank control.

Next, the plate was washed with PBS containing 0.05% Tween-20 (Amresco, Cat #M147). HRP-conjugated secondary antibody (Goat Anti-Mouse IgA, IgG (1, 2b, 2c, 3), IgM) (diluted 300-fold by 1% BSA, 0.1 mL per well) was added and incubated with the sample for 1 hour at 37° C. Next, the plate was washed and developed by adding 0.1 mL of TMB chromogen solution (Beyotime Biotechnology, Cat #P0209) to each well. After incubation in the dark at room temperature for 8 minutes, 0.1 mL of reaction solution (Beijing Dingguo Changsheng Biotechnology Co. LTD., Cat #EIA-0032) was added to each well. The optical absorption at 450 nm and 570 nm was measured using a microplate reader (Thermo MULTISKAN GO, Thermo Fisher Scientific), and the standard OD value was calculated.

The results showed that mice with humanized heavy chain immunoglobulin locus were capable of producing IgA, IgG1, IgG2b, IgG2c, IgG3 and IgM antibody isotypes, and the mice had similar expression levels for each isotype compared to wild-type mice (FIGS. 52-57 ). This indicated that humanization of the heavy chain variable region gene segments did not have a significant adverse effect on antibody class switching, the expression, or the secretion of the various antibody isotypes.

Example 11: V(D)J Recombination of Human Variable Region Gene Segments in Mice

The mRNA sequences of the heavy chain variable region and the light chain variable region in mice were analyzed by next generation sequencing.

One unimmunized (not exposed to a particular antigen) humanized heavy chain homozygous mouse was selected. Spleen cells were collected from the mouse for RNA extraction. A 5′ RACE kit (SMARTer RACE 5′/3′ Kit, Takara Bio USA, Inc., Cat #634858) was used to perform reverse transcription to obtain cDNA. The obtained cDNA was PCR-amplified using the IgM constant region-specific primer and the UPM primer of the 5′ RACE kit to obtain the heavy chain variable region sequence fragment, followed by sequencing. The IgM constant region-specific primer sequence was 5′-ccaagcttacgagggggaagacatttgggaa-3′ (SEQ ID NO: 50).

In another experiment, eleven light chain humanized heterozygous mice were selected, and RNA was extracted from retro-orbital blood. After reverse transcription by the method as described above, primers VKF1 and IgKC-tag were used to amplify the VκI family light chain genes, followed by sequencing. The following primer sequences were used:

VKF1 sequence: 5′-cataagatctcgmcatccrgwtgacccagt-3′ (SEQ ID NO: 51);

IgKC-tag primer sequence: 5′-ctaacactcattcctgttgaagctcttgac-3′ (SEQ ID NO: 52).

The sequencing results were compared to the NCBI Ig Blast tool for human immunoglobulin sequences to identify the expression of human V_(H), D_(H), J_(H) and V_(K), J_(K) genes after V(D)J recombination. In the 135 analyzed clones, preliminary results detected the expressions of certain V_(H), D_(H) gene segments and all J_(H) gene segments (Table 15). Some of these gene segments were located close to the modification site on the humanized fragment, and some were away from the modification site. This indicated that the human V_(H), D_(H) and J_(H) genes on the human chromosome fragment that were integrated into mice can be recombined to express the human heavy chain, after replacing the endogenous chromosome fragment with the human immunoglobulin heavy chain sequence.

Most of the VκI family light chain genes were detected in the 441 clones derived from humanized light chain heterozygous mice (Table 16). Similar to the heavy chain detection results, some of these genes on humanized fragment were located very close to the modification site, and some were away from the modification site. This indicated that after replacing the endogenous light chain immunoglobulin variable region locus with the human light chain immunoglobulin variable region locus, the human Vκ and Jκ genes that were integrated into the mouse genome can be recombined to express light chain with human light chain variable region. Further analysis of the results in the eleven mice showed that the detected distribution of the IGKV genes was not significantly different among the tested mice (FIG. 58 ).

TABLE 15 List of detected IGHV genes, IGHD genes, and IGHJ that were expressed after VDJ recombination Observed Observed Observed value value IGHJ value IGHV gene (count) IGHD gene (count) gene (count) IGHV1-18 1 IGHD1-1 4 IGHJ1 2 IGHV1-24 1 IGHD1-20 2 IGHJ2 16 IGHV1-46 2 IGHD1-26 8 IGHJ3 7 IGHV1-69 1 IGHD1-7 10 IGHJ4 55 IGHV2-70 1 IGHD2-2 2 IGHJ5 12 IGHV3-15 1 IGHD2-21 2 IGHJ6 43 IGHV3-21 5 IGHD3-10 5 IGHV3-23 7 IGHD3-16 3 IGHV3-30 10 IGHD3-22 3 IGHV3-30-3 2 IGHD3-3 2 IGHV3-33 6 IGHD3-9 1 IGHV3-43 7 IGHD4-11 4 IGHV3-48 5 IGHD4-17 7 IGHV3-49 1 IGHD4-23 1 IGHV3-66 1 IGHD5-12 4 IGHV3-73 1 IGHD5-18 1 IGHV3-74 3 IGHD5-24 1 IGHV4-30-4 5 IGHD6-13 17 IGHV4-34 37 IGHD6-19 22 IGHV4-39 14 IGHD6-25 1 IGHV4-4 1 IGHD6-6 5 IGHV4-59 11 IGHD7-27 14 IGHV5-51 1 IGHV6-1 11

TABLE 16 List of detected IGKV genes that were expressed after VJ recombination Gene names Observed value (count) IGKV1D-43 9 IGKV1D-13 4 IGKV1D-16 9 IGKV1D-12 11 IGKV1-39 17 IGKV1-37 2 IGKV1-33 107 IGKV1-27 14 IGKV1-17 55 IGKV1-16 26 IGKV1-13 1 IGKV1-12 50 IGKV1-9 42 IGKV1-8 24 IGKV1-6 31 IGKV1-5 31 IGKV4-1 8

Example 12: Immunization and Antibody Production in Humanized Mice

Five wild-type (WT) mice and five humanized heavy chain homozygous mice (9-10 week old) were randomly selected and immunized with exogenous antigens. The mice were repeatedly immunized once every two weeks for a total of three immunizations. Retro-orbital blood was collected after the second immunization and the third immunization. Serum was collected and then the serum titer was measured by ELISA or FACS to determine and analyze the antigen-specific antibody response. Three antigens were used in the study, which were hBTLA, dPD1 and OVA (FIGS. 59-63 ). The results showed that after the second immunization, most wild-type (WT) and humanized heavy chain homozygous mice produced antigen-specific antibodies. After the third immunization, the antibody titer increased to 1×10⁴ to 1×10⁵. The immunopotency test results were essentially the same in humanized mice as compared to the wildtype mice, indicating that the humanized immunoglobulin variable region locus in the mice are functional and can produce antigen-specific antibodies.

Example 13: B-Cell Development in hVH/hVL Mice

The mice with homozygous humanized heavy chain immunoglobulin locus (humanized VH mice or hVH mice) and the mice with humanized light chain immunoglobulin locus (humanized VL mice or hVL mice) were crossed with each other to obtain mice with both homozygous humanized heavy chain immunoglobulin locus and homozygous humanized light chain immunoglobulin locus (humanized VH/VL mice, or hVH/hVL mice). The hVH/hVL mice can be used produce humanized monoclonal antibody in vivo.

Experiments were performed to compare the immune systems of the naïve humanized VH/VL mice and the naïve wild-type mice. The body weight and spleen weight were measured in wild-type and hVH/hVL mice (FIGS. 65A-65B). No significant differences in average body weight and spleen weight were detected between wild-type and hVH/hVL mice.

Flow cytometry was performed to analyze lymphocyte populations and distribution in the spleen (FIG. 66 ) and B cell populations in the spleen and bone marrow (FIGS. 67A-67B, 68A-68C) of the mice. The results showed that in hVH/hVL mice, the percentage of B cells, T cells, NK cells, CD4+ T cells and CD8+ T cells in spleen were almost identical to those of wild type mice. In the results, the leukocytes included: B cells (e.g., characterized by CD45+, CD19+, TCR−), T cells, and natural killer (NK) cells (e.g., characterized by CD45+, TCR−, NK1.1+). T cells were characterized by CD45+, CD19−, TCR+. CD4+ T Cells (CD4) were characterized by CD45+, CD19−, TCR+, CD4+, CD8−. And CD8+ T cells (CD8) were characterized by CD45+, CD19−, TCR+, CD4−, CD8+. Only intact, single, live leukocytes were included in the flow cytometry analysis.

FIG. 67A indicated percentages of B cells at different developmental stages. The development stages of B cells in spleen were categorized into T1 (Transitional type 1 B cell, characterized by B220⁺IgM⁺IgD⁻), T2 (Transitional type 2 B cell, characterized by B220⁺IgM⁺IgD⁻) and mature B cells (characterized by B220⁺IgM^(low)IgD⁺). In addition, B cell development were also evaluated at the spleen marginal zone (Marginal-zone B cell, MZ-B, characterized by B220⁺CD21⁺CD23⁻) and follicular zone (Follicular B cell, referred to as FO-B, characterized by B220+CD21^(low)CD23⁺). FIG. 67B showed percentages of splenic B cells at spleen marginal zone (MZ-B) and follicular zone (FO-B). No significant differences were observed between the wild-type mice and hVH/hVL mice.

FIG. 68A indicated percentages of B cells at different developmental stages in bone marrows. B cell progenitor cells in bone marrow were analyzed by flow cytometry. Based on expression levels of B220 and CD43, B cell progenitor cells in bone marrow can be divided into 3 cell populations pro-B-cells (characterized by B220^(low)CD43^(high)IgM^(low)), pre-B-cells (characterized by B220^(low)CD43^(int)IgM^(low)) and immature-B-cells (characterized by B220^(high)CD43^(low)IgM^(high)). No significant differences were observed between the wild-type mice and hVH/hVL mice.

In addition, B cell development were also evaluated in bone marrow or spleen by flow cytometry to selectively stain plasma cells (B220^(low)IgM⁻IgD⁻CD138⁻) and memory B cells (B220⁺IgM⁺IgD⁻CD38⁺) (FIGS. 68B-68C). No significant difference was observed between the wild-type mice and hVH/hVL mice.

Different immunoglobulin (Ig) subtypes in the serum of hVH/hVL and wild-type mice were quantitatively measured by ELISA. A total of six mice were selected for each group. No significant differences in IgA, IgG1, IgG2b, IgG2c, IgG3 and IgM levels were observed (FIG. 69 ).

These experiments showed that the immune system in the hVH/hVL mice is functional and the humanized immunoglobulin locus in hVH/hVL mice can properly interact with the mouse immunoglobulin constant regions.

Example 14: Analysis of Germline Usage in hVH/hVL Mice

The heavy chain IGHV, IGHD and IGHJ usage in the hVH/hVL naïve mice (without antigen stimulation) was analyzed. The results are shown in FIGS. 70A-70D. In addition, the kappa chain IGKV and IGKJ usage was also analyzed. The results are shown in FIGS. 71A-71C.

Germline usage in naive hVH/hVL mice was determined by next generation sequencing (NGS). For example, as shown in FIG. 71C, IGKJ1, IGKJ2 and IGKJ4 were frequently used in naive hVH/hVL mice, while IGKJ3 and IGKJ5 were less frequently observed. Such an IGKJ germline usage pattern is consistent with literature reports of human IGKJ germline usage.

The heavy chain CDR3 length distribution was determined by NGS sequencing of immune repertoire from the splenocytes of naive hVH/hVL mice (n=2). As shown in FIG. 72 , the median length of CDR3 was 14 amino acids. The results were consistent with the median length of human heavy chain CDR3 in the human immune system.

The type of amino acids at each position of heavy chain CDR3 (HCDR3) was analyzed (FIG. 73 ). Multiple patterns, including the increasing frequency of tyrosine usage and the increasing usage of DH2 (IGHD2) germline family, were observed. These patterns are similar to the amino acid composition in human HCDR3.

Cysteine residue can form disulfide bond. Human HCDR3 may contain one cysteine residue or two cysteine residues, while mouse HCDR3 typically contains no cysteine. Results in FIG. 74 shows that the frequency of HCDR3 of the hVH/hVL mice that contains cysteine residues and the frequency increases as the length of HCDR3 increases. This result is consistent with the HCDR3 diversity in human peripheral blood mononuclear cells (PBMCs).

Example 15: Lymphoid Organ Histology Analysis

Spleen, inguinal lymph node, and Peyer's patch from naïve wild-type or naïve hVH/hVL mice were stained with H&E. Representative sections are shown in FIGS. 75A-75F. Wild-type (C57BL/6) mice and hVH/hVL mice exhibited normal structure with well-defined follicles and no significant differences in histological morphology were observed.

Example 16: Antibody Generation in hVH/hVL Mice

After the second and the third immunization with BCMA, IL4R, PD-1, Siglec15, and SIRPα antigens, blood was collected and the antigen-specific antibody titers of wild-type (C57BL6) mice and hVH/hVL mice were analyzed by ELISA (FIGS. 76A-76E). The results showed that hVH/hVL mice can produce antibodies that specifically binds to the antigen and the immune response of the wild-type and hVH/hVL mice were similar.

Example 17: B-Cell Development in hVH/hVL Mice

Experiments were performed to compare the immune systems of the humanized VH/VL mice and the wild-type mice after immunization. The body weight and spleen weight were measured in wild-type and hVH/hVL mice (FIGS. 77A-77B). No significant differences in average body weight and spleen weight were detected between wild-type and hVH/hVL mice.

Flow cytometry was performed to analyze lymphocyte populations and distribution in the spleen (FIG. 78 ) and B cell populations in the spleen and bone marrow (FIGS. 79A-79B, 80A-80C) of the mice.

FIG. 79A indicated percentages of B cells at different developmental stages in spleen. In addition, B cell development were also evaluated at the spleen marginal zone and follicular zone. FIG. 79B showed percentages of splenic B cells at spleen marginal zone (MZ-B) and follicular zone (FO-B). No significant differences were observed between the wild-type mice and the hVH/hVL mice.

FIG. 80A indicated percentages of B cells at different developmental stages in bone marrows. B cell progenitor cells in bone marrow were analyzed by flow cytometry. No significant differences were observed between the wild-type mice and the hVH/hVL mice.

In addition, B cell development were also evaluated in bone marrow or spleen by flow cytometry to selectively stain plasma cells (B220^(low)IgM⁻IgD⁻CD138⁻) and memory B cells (B220⁺IgM⁺IgD⁻CD38⁺) (FIGS. 80B-80C). No significant differences were observed between the wild-type mice and the hVH/hVL mice.

Different immunoglobulin (Ig) subtypes in the serum of hVH/hVL and wild-type mice were quantitatively measured by ELISA. A total of six mice were selected for each group. No significant differences in IgA, IgG1, IgG2b, IgG2c, IgG3 and IgM levels were observed (FIG. 81 ). In addition, the total amount of IgG in the serum of hVH/hVL and wild-type mice were quantitatively measured by ELISA. No significant differences were observed (FIG. 82 ).

Other Embodiments

It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims. 

What is claimed is:
 1. A method of making a genetically-modified rodent, the method comprising: (a) modifying a first human chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different, wherein the first human chromosome is human chromosome 14; (b) modifying a first endogenous rodent chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different; (c) introducing the modified first human chromosome into a cell of the rodent; and (d) inducing site specific recombination between the modified first human chromosome and the modified first endogenous chromosome, thereby replacing a sequence at the endogenous heavy chain immunoglobulin gene locus with a sequence from the modified first human chromosome, wherein the sequence from the modified first human chromosome comprises at least human IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76, IGHV3-75, and IGHV(II)-74-1, wherein the sequence from the modified first human chromosome is integrated into the endogenous chromosome by one recombination step, and the integrated sequence from the one recombination step at the endogenous heavy chain immunoglobulin gene locus is at least 500 kb, wherein an offspring of the rodent can produce a humanized antibody.
 2. The method of claim 1, wherein (1) at least 150 human IGHV genes selected from the group consisting of IGHV(III)-82, IGHV3-57, IGHV3-36, IGHV3-23, IGHV1-69, IGHV(IV)-44-1, IGHV3-30-22, IGHV3-11, IGHV7-81, IGHV7-56, IGHV3-35, IGHV(III)-22-2, IGHV1-68, IGHV(III)-44, IGHV(II)-30-21, IGHV2-10, IGHV4-80, IGHV4-55, IGHV7-34-1, IGHV(II)-22-1, IGHV(III)-67-4, IGHV(II)-43-1, IGHV4-30-2, IGHV3-9, IGHV3-79, IGHV3-54, IGHV4-34, IGHV3-22, IGHV(III)-67-3, IGHV3-43, IGHV4-30-1, IGHV1-8, IGHV(II)-78-1, IGHV(II)-53-1, IGHV3-33-2, IGHV3-21, IGHV(III)-67-2, IGHV3-42, IGHV3-30-2, IGHV5-10-1, IGHV5-78, IGHV3-53, IGHV(II)-33-1, IGHV(II)-20-1, IGHV(II)-67-1, IGHV3-41, IGHV(II)-30-1, IGHV3-64D, IGHV7-77, IGHV3-52, IGHV3-33, IGHV3-20, SLC20A1P1 (GLVR1), IGHV(II)-40-1, IGHV3-30, IGHV3-7, IGHV(III)-76-1, IGHV(II)-51-2, GOLGA4P1, IGHV3-19, IGHV1-67, IGHV7-40, GOLGA4P2, IGHV3-6, IGHV3-76, IGHV(III)-51-1, IGHV3-32, IGHV1-18, IGHV3-66, IGHV4-39, IGHV3-29, IGHV(III)-5-2, IGHV3-75, IGHV5-51, IGHV(II)-31-1, SLC20A1P2, IGHV(II)-65-1, IGHV1-38-4, IGHV(II)-28-1, IGHV(III)-5-1, IGHV(II)-74-1, IGHV3-50, IGHV4-31, IGHV1-17, IGHV3-65, IGHV(III)-38-1D, IGHV4-28, IGHV2-5, IGHV3-74, IGHV(II)-49-1, IGHV3-30-52, IGHV(III)-16-1, IGHV3-64, IGHV3-38-3, IGHV7-27, IGHV7-4-1, IGHV3-73, IGHV3-49, IGHV(II)-30-51, IGHV3-16, GOLGA4P3, IGHV(III)-44D, IGHV(II)-26-2, IGHV4-4, IGHV3-72, IGHV3-48, IGHV3-30-5, IGHV(II)-15-1, IGHV3-63, IGHV(II)-43-1D, IGHV(III)-26-1, IGHV1-3, IGHV3-71, IGHV(III)-47-1, IGHV3-30-42, IGHV3-15, IGHV(II)-62-1, IGHV3-43D, IGHV2-26, IGHV(III)-2-1, IGHV2-70, IGHV3-47, IGHV(II)-30-41, IGHV1-14, IGHV3-62, IGHV3-42D, IGHV(III)-25-1, IGHV1-2, IGHV1-69D, IGHV(II)-46-1, IGHV4-30-4, IGHV(III)-13-1, IGHV4-61, IGHV7-40D, IGHV3-25, IGHV1-69-2, IGHV1-46, IGHV3-30-33, IGHV3-13, IGHV(II)-60-1, IGHV4-38-2, IGHV1-24, IGHV3-69-1, IGHV1-45, IGHV(II)-30-32, IGHV1-12, IGHV3-60, IGHV(III)-38-1, IGHV3-23D, IGHV(II)-1-1, IGHV2-70D, IGHV(II)-44-2, IGHV3-30-3, IGHV(III)-11-1, IGHV4-59, IGHV3-38, IGHV(III)-22-2D, IGHV6-1, IGHV1-58, IGHV3-37, and IGHV(II)-22-1D are integrated into the endogenous chromosome by recombination; (2) at least 20 human IGHD genes selected from the group consisting of IGHD1-1, IGHD2-8, IGHD2-15, IGHD3-22, IGHD2-2, IGHD3-9, IGHD3-16, IGHD4-23, IGHD3-3, IGHD3-10, IGHD4-17, IGHD5-24, IGHD4-4, IGHD4-11, IGHD5-18, IGHD6-25, IGHD5-5, IGHD5-12, IGHD6-19, IGHD1-26, IGHD6-6, IGHD6-13, IGHD1-20, IGHD1-7, IGHD1-14, IGHD2-21, and IGHD7-27 are integrated into the endogenous chromosome by recombination; and (3) at least 5 human IGHJ genes selected from the group consisting of IGHJ1P, IGHJ1, IGHJ2, IGHJ2P, IGHJ3, IGHJ4, IGHJ5, IGHJ3P, and IGHJ6 are integrated into the endogenous chromosome by recombination.
 3. The method of claim 1, the method further comprising: (e) modifying a second human chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different; (f) modifying a second endogenous rodent chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different; (g) introducing the modified second human chromosome into a cell of the rodent; and (h) inducing site specific recombination between the modified second human chromosome and the modified second endogenous chromosome, thereby replacing a sequence at the endogenous light chain immunoglobulin gene locus with a sequence from the modified second human chromosome, wherein the sequence from the modified second human chromosome is integrated into the endogenous chromosome by one recombination step, and the sequence from the modified second human chromosome is at least 500 kb.
 4. The method of claim 3, wherein the method comprises replacing a sequence at the endogenous light chain immunoglobulin gene locus with a sequence comprising at least 50 human IGKV genes selected from the group consisting of IGKV3D-7, IGKV3D-25, IGKV2-36, IGKV1-16, IGKV1D-16, IGKV1D-35, IGKV2-26, IGKV1-6, IGKV3D-11, IGKV2D-30, IGKV3-31, IGKV3-11, IGKV3D-20, IGKV2D-40, IGKV6-21, IGKV4-1, IGKV1D-8, IGKV2D-26, IGKV1-35, IGKV3-15, IGKV1D-17, IGKV2D-36, IGKV3-25, IGKV1-5, IGKV1D-12, IGKV3D-31, IGKV2-30, IGKV2-10, IGKV6D-21, IGKV2-40, IGKV3-20, IGKV1D-43, IGKV1D-27, IGKV3-34, IGKV2-14, IGKV6D-41, IGKV1D-37, IGKV2-24, IGKV2-4, IGKV1D-13, IGKV1D-32, IGKV2-29, IGKV1-9, IGKV1D-22, IGKV1-39, IGKV2-19, IGKV1D-42, IGKV2D-28, IGKV1-33, IGKV1-13, IGKV2D-18, IGKV2D-38, IGKV2-23, IGKV7-3, IGKV2D-14, IGKV1D-33, IGKV2-28, IGKV1-8, IGKV2D-23, IGKV2-38, IGKV2-18, IGKV2D-10, IGKV2D-29, IGKV1-32, IGKV1-12, IGKV2D-19, IGKV1D-39, IGKV1-22, IGKV5-2, IGKV3D-15, IGKV3D-34, IGKV1-27, IGKV3-7, IGKV2D-24, IGKV1-37, and IGKV1-17, and at least 3 human IGKJ genes selected from the group consisting of IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5.
 5. The method of claim 3, wherein the rodent comprises the entire human IGKV genes and the entire human IGKJ genes at the endogenous light chain immunoglobulin gene locus.
 6. The method of claim 3, wherein the sequence from the modified second human chromosome is at least 1000 kb.
 7. The method of claim 3, wherein the rodent is capable of expressing IGKV1D-12, IGKV2D-30, IGKV1-27, IGKV6-21, IGKV1-6, IGKV1-39, IGKV1-16, IGKV1-17, IGKV2D-29, IGKV3-11, IGKV1-5, IGKV1-12, IGKV1-9, IGKV3-20, IGKV3-15, IGKV2-28, IGKV2-30, IGKV1-33, IGKV4-1, IGKV6D-41, IGKV3D-11, IGKV2D-24, IGKV1D-43, IGKV1D-17, IGKV7-3, IGKV2D-26, IGKV1-37, IGKV2-29, IGKV1-13, IGKV5-2, IGKV2-40, IGKV3-7, IGKV3D-20, IGKV1D-8, IGKV6D-21, IGKV1-8, IGKV2-24, and IGKV3D-15.
 8. The method of claim 1, wherein the rodent has a complete human antibody repertoire.
 9. The method of claim 1, wherein the rodent is capable of expressing IGHV3-15, IGHV3-53, IGHV3-66, IGHV5-51, IGHV1-24, IGHV1-18, IGHV1-69, IGHV3-7, IGHV3-74, IGHV3-23, IGHV3-43, IGHV3-21, IGHV3-30-3, IGHV4-59, IGHV6-1, IGHV3-48, IGHV4-34, IGHV4-39, IGHV3-33, IGHV3-30, IGHV4-61, IGHV3-47, IGHV3-NL1, IGHV3-69-1, IGHV7-4-1, IGHV1-58, IGHV4-28, IGHV2-26, IGHV2-5, IGHV3-64, IGHV3-20, IGHV2-70, IGHV3-11, IGHV4-30-2, IGHV3-13, IGHV3-49, IGHV5-10-1, IGHV3-72, IGHV1-2, IGHV4-30-4, IGHV1-46, IGHV3-64D, IGHV1-3, IGHV3-73, IGHV4-4, and IGHV4-31.
 10. The method of claim 1, wherein the rodent is a mouse.
 11. A method of making a genetically-modified mouse, the method comprising: (a) modifying a first human chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different, (b) modifying a first endogenous mouse chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different; (c) introducing the modified first human chromosome into a cell of the mouse; and (d) inducing site specific recombination between the modified first human chromosome and the modified first endogenous chromosome, thereby replacing a sequence at the endogenous heavy chain immunoglobulin gene locus with a sequence from the modified first human chromosome, thereby replacing, at an endogenous heavy chain immunoglobulin locus, a sequence starting from mouse IGHV1-85 gene to mouse IGHJ4 gene with an exogenous heavy chain immunoglobulin locus sequence comprising human IGHV(III)-82, IGHV7-81, IGHV4-80, IGHV3-79, IGHV(II)-78-1, IGHV5-78, IGHV7-77, IGHV(III)-76-1, IGHV3-76, IGHV3-75, and IGHV(II)-74-1, wherein the exogenous heavy chain immunoglobulin locus sequence is operably linked to one or more of endogenous IGHM, IGHδ, IGHG, IGHE, and IGHA genes, wherein the exogenous heavy chain immunoglobulin locus sequence is integrated into the endogenous heavy chain immunoglobulin locus by one recombination step, wherein the exogenous heavy chain immunoglobulin locus sequence is at least 500 kb, wherein an offspring of the mouse can produce a humanized antibody.
 12. The method of claim 11, wherein the mouse has a complete human antibody repertoire.
 13. A method of making a genetically-modified rodent, the method comprising: (a) modifying a first human chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different; (b) modifying a first endogenous rodent chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different; (c) introducing the modified first human chromosome into a cell of the rodent; and (d) inducing site specific recombination between the modified first human chromosome and the modified first endogenous chromosome, thereby replacing a sequence at the endogenous light chain immunoglobulin gene locus with a sequence from the modified first human chromosome, wherein the sequence from the modified first human chromosome comprises one or more human IGKV genes and one or more human IGKJ genes, wherein the sequence from the modified first human chromosome is integrated into the endogenous light chain immunoglobulin gene locus by one recombination step, and the sequence from the modified first human chromosome is at least 500 kb, wherein an offspring of the rodent can produce a humanized antibody.
 14. The method of claim 13, the method further comprising: (e) modifying a second human chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different; (f) modifying a second endogenous rodent chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different; (g) introducing the modified second human chromosome into a cell of the rodent; and (h) inducing site specific recombination between the modified second human chromosome and the modified second endogenous chromosome, thereby replacing a sequence at the endogenous heavy chain immunoglobulin gene locus with a sequence from the modified second human chromosome, wherein the sequence from the modified second human chromosome is integrated into the endogenous chromosome by one recombination step, and the sequence from the modified second human chromosome is at least 500 kb.
 15. The method of claim 13, wherein the method comprises replacing at the endogenous light chain immunoglobulin gene locus, a sequence starting from mouse IGKV2-137 gene to mouse IGKJ5 gene with a sequence comprising at least 50 human IGKV genes selected from the group consisting of IGKV3D-7, IGKV3D-25, IGKV2-36, IGKV1-16, IGKV1D-16, IGKV1D-35, IGKV2-26, IGKV1-6, IGKV3D-11, IGKV2D-30, IGKV3-31, IGKV3-11, IGKV3D-20, IGKV2D-40, IGKV6-21, IGKV4-1, IGKV1D-8, IGKV2D-26, IGKV1-35, IGKV3-15, IGKV1D-17, IGKV2D-36, IGKV3-25, IGKV1-5, IGKV1D-12, IGKV3D-31, IGKV2-30, IGKV2-10, IGKV6D-21, IGKV2-40, IGKV3-20, IGKV1D-43, IGKV1D-27, IGKV3-34, IGKV2-14, IGKV6D-41, IGKV1D-37, IGKV2-24, IGKV2-4, IGKV1D-13, IGKV1D-32, IGKV2-29, IGKV1-9, IGKV1D-22, IGKV1-39, IGKV2-19, IGKV1D-42, IGKV2D-28, IGKV1-33, IGKV1-13, IGKV2D-18, IGKV2D-38, IGKV2-23, IGKV7-3, IGKV2D-14, IGKV1D-33, IGKV2-28, IGKV1-8, IGKV2D-23, IGKV2-38, IGKV2-18, IGKV2D-10, IGKV2D-29, IGKV1-32, IGKV1-12, IGKV2D-19, IGKV1D-39, IGKV1-22, IGKV5-2, IGKV3D-15, IGKV3D-34, IGKV1-27, IGKV3-7, IGKV2D-24, IGKV1-37, and IGKV1-17, and at least 3 human IGKJ genes selected from the group consisting of IGKJ1, IGKJ2, IGKJ3, IGKJ4, and IGKJ5.
 16. The method of claim 13, wherein the sequence from the modified second human chromosome is at least 1000 kb.
 17. The method of claim 13, wherein the rodent has a complete human antibody repertoire.
 18. The method of claim 13, wherein the rodent is a mouse.
 19. A method of making a genetically-modified rodent, the method comprising: (a) modifying a human chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different; (b) modifying an endogenous rodent chromosome to introduce a first recombination site and a second recombination site, wherein the first recombination site and the second recombination site are different; (c) introducing the modified human chromosome into a cell of the rodent; and (d) inducing recombination between the modified human chromosome and an endogenous chromosome, thereby replacing a sequence at the endogenous chromosome with a sequence from the modified human chromosome, wherein the sequence from the modified human chromosome is integrated into the endogenous chromosome by one recombination step, and the integrated sequence from the one recombination step at an endogenous gene locus of interest is at least 500 kb. 